Analytical, Nutritional and Clinical MethodsRaman spectroscopic determination of extent of O-esterification in acetylated soy protein isolates
Introduction
Soy protein isolates (SPI) are widely used by the food industry as functional ingredients in the manufacturing of many processed foods. In the food industry, chemical modification is often performed to expand the range of protein functional properties. Acetylation with acetic anhydrides has been shown to be a very powerful tool for improving the functional properties of SPI (Franzen & Kinsella, 1976a; Umeya, Mitsuishi, Yamauchi, & Shibasaki, 1981) and many plant proteins, including leaf protein (Franzen & Kinsella, 1976b), sunflower (Kabirrullah & Wills, 1982), cottonseed (Rahma & Narasinga Rao, 1983), winged bean (Narayana & Narasinga Rao, 1984), oat (Ma, 1984), faba bean (Muschiolik, Dickinson, Murray, & Stainsby, 1987), rapeseed (Gruener & Ismond, 1997), chickpea (Liu & Hung, 1998), and mung bean (Ei-Adaway, 2000). The related changes in functional and physicochemical properties of food proteins are affected by the degree of modification (Schwenke, 1997). Although the ε-amino group of lysine is the most readily acylated group in proteins, the acylating reagents can also react with other nucleophilic groups, such as phenolic (tyrosine) and aliphatic (serine and threonine) hydroxyl groups (Howell, 1996). The level of acylation (esterification) of hydroxy amino acids increases sharply when the reaction with the lysine is essentially complete (Fraenkel-Conrat, 1959; Gounaris & Perlamnn, 1967; Schwenke, Zirwer, Gast, Gornitz, Linow, & Gueguen, 1990). Furthermore, it has been documented that drastic conformational changes occurred with the appearance of a large amount of O-esterification, which contributes markedly to changes in charge and hydrophobicity (Schwenke, Mothes, Zirwer, Gueguen, & Subirade, 1993a; Schwenke, Dudek, mothes, Raab, & Seifert, 1993b). Therefore, it is important to measure the level of O-esterification to control quantitatively the desired degree of acetylation of a protein for use in different processes.
Traditionally, the extent of O-esterification in chemically modified proteins is measured using wet chemistry techniques (Habeeb & Atassi, 1969; Hall, Trinder, & Givens, 1973; Hestrin, 1949). These methods are destructive, involve time-consuming sample preparation procedures, and require relatively large amounts of samples. Furthermore, the wet chemistry methods are not amenable to continuous monitoring for quality control. Thus, it would be most convenient to select an analytical method that could use solid proteins without any need for sample preparation as well as to provide a direct, non-destructive, and faster determination of the degree of acetylation in the modified proteins.
Recently, Phillips et al., 1999a, Phillips et al., 1999b developed an analytical method using Raman spectroscopy for the determination of degree of acetylation and succinylation in modified starches. Raman spectroscopy can analyze samples directly, in air, at ambient temperature and pressure, wet or dry, and in many cases without destroying the sample. The intensity of a Raman band contributing to a Raman spectrum depends linearly on the amount of substance contributing to that Raman band (Hendra, Jones, & Warnes, 1991; Wetzel & LeVin, 1999). Raman spectroscopy has been used as a quantitative analytical tool in the pharmaceutical and polymer industries (Hendra et al., 1991) and its potential in food analysis has been demonstrated (Davies, Binns, Melia, & Bourgeois, 1990; Ma & Phillips, 2002; Ozaki, Cho, Ikegaya, Muraishi, & Kawauchi, 1992; Sadeghi-Jorbachi, Wilson, & Belton, 1991; Thygesen, Lokke, Micklander, & Engelsen, 2003).
In this paper, we describe the development of an analytical technique based on Raman spectroscopy to determine the extent of O-esterification in acetylated SPI. Esterification of hydroxy amino acids by acetic anhydride appends ester carbonyl groups to the protein and these groups contain CO bonds that exhibit a characteristic Raman vibration at around 1737 cm−1. We will demonstrate that the intensity of this CO vibrational band has a linear relationship with the extent of O-esterification of the soy protein, and the band is sufficiently intense to be conveniently used for quick and accurate determination of the extent of O-esterification in proteins. To our knowledge, this is the first reported use of Raman spectroscopy to determine the extent of O-esterification of chemically modified SPI.
Section snippets
Acetylation
Commercial SPI, Supro 610, were obtained from Protein Technology International (St. Louis, MO). It was acetylated according to the procedure described by Franzen and Kinsella (1976a) with some modifications of the procedure. A dispersion (≈2.5%, w/v) of SPI was prepared by mixing 2 g of protein sample with approximately 80 ml distilled water followed by 1 h magnetic stirring. The pH of the dispersion was adjusted to about 8.0 by adding 2 N sodium hydroxide. Acetylation was accomplished by the
Results and discussion
The extent of modification of the functional groups of a protein can be varied widely by changing the amount of the acylating agent employed. O-esterification occurs at a sufficiently high excess of the reagent (Fraenkel-Conrat, 1959; Gounaris & Perlamnn, 1967; Schwenke et al., 1990). The Raman spectra of the control non-acetylated SPI sample and four acetylated SPI samples with different levels of modification are shown in Fig. 1. Assignment of some major Raman bands was made based on results
Conclusions
The Raman spectroscopic method presented in this paper has several important advantages over other methods for the determination of level of esterification in modified proteins. The Raman method requires almost no sample preparation for the solid soy proteins which makes the method much more convenient as well as potentially faster. The technique is non-destructive whereas the wet chemistry methods are destructive tests usually requiring toxic and/or corrosive reagents. The Raman method also
Acknowledgments
Financial supports were received from a Hong Kong University Conference and Research Grants Committee grant to CYM, Hong Kong Research Grant Council awards to CYM and DLP, and a Faculty Collaborative Research grant to CYM and DLP.
References (35)
- et al.
Fourier transform Raman spectroscopy of polymeric biomaterials and drug delivery systems
Spectrochimica Acta A
(1990) - et al.
Effect of acetylation and succinylation on functional properties of the canola 12S globulin
Food Chemistry
(1997) - et al.
Succinylation of pepsinogen
The Journal of Biological Chemistry
(1967) - et al.
Enzymic and immunochemical properties of lysozyme-II
Immunochemistry
(1969) The application of Raman spectroscopy in food science
Trends in Food Science Technology
(1996)Raman spectroscopy of DNA and proteins
Methods in Enzymology
(1995)- et al.
Vibrational microspectroscopy of food. Raman vs. FT-IR
Trends in Food Science Technology
(2003) - et al.
Introduction to infrared and Raman spectroscopy
(1990) Functional properties and nutritional quality of acetylated and succinylated mung bean protein isolate
Food Chemistry
(2000)Methods for investigating the essential groups for enzyme activity
Methods in Enzymology
(1959)
Functional properties of succinylated and acetylated soy proteins
Journal of Agricultural and Food Chemistry
Functional properties of succinylated and acetylated leaf protein
Journal of Agricultural and Food Chemistry
Observations on the use of 2,4,6-trinitrobenzenesulphonic acid for the determination of available lysine in animal protein concentrates
Analyst
Fourier transform Raman spectroscopy, instrumentation and chemical applications
Acylation reactions mediated by purified acetylcholine esterase
The Journal of Biological Chemistry
Chemical and enzymatic modifications
Functional properties of acetylated and succinylated sunflower protein isolate
Journal of Food Technology
Cited by (20)
Spectroscopic analysis of the effect of vitamin B<inf>12</inf>-soy protein isolate on the soy protein isolate structure
2021, Journal of Molecular LiquidsCitation Excerpt :It was found that the ratio of 1780 cm−1 band strength to 1003 cm−1 band strength of C-O stretching vibration increased with the increase of the deamidation degree of SPI. Yu et al. [17] used Raman spectroscopy to analyze the esterification effect and structural change law of SPI with the acetyl group. It was found that the relative absorption intensity of the peak near 1737 cm−1 was proportional to the degree of O-esterification.
Fungal fermentation inducing improved nutritional qualities associated with altered secondary protein structure of soybean meal determined by FTIR spectroscopy
2020, Measurement: Journal of the International Measurement ConfederationCitation Excerpt :Unfermented SBM has a predominant protein confirmation of β-sheet of 40.76%, and this was followed by a 47.00% of β-turn, 19.16% α-helix with a 47% ratio of α-helix:β-sheet and no random coil. Relatively comparable results for SBM were previously reported [74–76]. In detailed examination of FSD spectra pattern of fermented SBM samples in Fig. 2, it was seen that the proportion of secondary protein components in fungal fermented SBM were different from unfermented SBM.
Improvement of nutritional quality of soybean meal by Fe(II)-assisted acetic acid treatment
2019, Food ChemistryCitation Excerpt :The untreated SPI contained 15.33% α-helix, 44.70% β-sheet, 24.28% β-turn and 15.68% random coils. The results showed that the predominant conformation in SPI is β-sheet, which confirmed the results of previous studies (Rampon, Robert, Nicolas, & Dufour, 1999; Yu, Ma, Yuen, & Phillips, 2004). We observed that different treatments gently changed the secondary structural of SPI.
Raman spectroscopic determination of structural changes in meat batters upon soy protein addition and heat treatment
2008, Food Research InternationalCHAPTER 8: Protein Modifications and the Food Matrix: Consequences, Chemistry and Characterization
2021, Food Chemistry, Function and Analysis