改性水溶性大豆多糖对鸡蛋涂膜保鲜效果的比较研究
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摘要
水溶性大豆多糖（soluble soybean polysaccharide，SSPS）是一种从大豆子叶中提取的水溶性酸性多糖，分子量范围在5000～1000000之间。它具有多方面的优良性质，可以作为高膳食纤维，具有高热、酸、盐稳定性，乳化性，低粘性，抑菌性及优良的胶着性和成膜性。因此，基于人们对食品健康、安全的要求，可以将水溶性大豆多糖作为洁蛋生产工艺中的鸡蛋涂膜保鲜材料使用。
目前，已经有前人研究水溶性大豆多糖对鸡蛋的涂膜保鲜效果，但是效果不甚理想。这是由于多糖一些自身结构的限制，使得多糖制成的涂膜保鲜剂的抗菌性和抗水性较弱。而且前人研究的水溶性大豆多糖涂膜保鲜剂中水溶性大豆多糖和其它添加剂的使用量大，对鸡蛋的保鲜效果也不是特别明显。
因此，本试验主要研究水溶性大豆多糖的物理和化学改性，并且选择合适的添加剂配制成鸡蛋涂膜保鲜剂，对鸡蛋进行涂膜，研究其保鲜效果。同时，初步探究其对鸡蛋涂膜保鲜的机理。具体研究结果如下：
（1）采用弱酸高温热提取法从湿豆渣中提取粗制的水溶性大豆多糖，提取率为34.2%。然后分别用乙醇沉淀法和超滤法分离、纯化粗制的水溶性大豆多糖，其结果为：乙醇沉淀法的多糖得率和脱色率分别为31.4%和92.1%，超滤法的多糖得率和脱色率分别为33.9%和90.6%。通过超滤法还得到了不同相对分子质量的水溶性大豆多糖，并且超滤法还具有一定的浓缩效果。
（2）将水溶性大豆多糖经物理改性后得到高相对分子质量水溶性大豆多糖（H-M）和低相对分子质量水溶性大豆多糖（L-M）。分别以浓度为3.0%（w/v）的分离纯化的SSPS、H-M和L-M为主要原料，浓度为0.5%（w/v）的海藻酸钠作为增稠剂，浓度为0.6%（w/v）的山梨酸钾作为辅助抑菌剂，配制成不同的鸡蛋涂膜保鲜剂对鸡蛋进行涂膜保鲜试验。在高温、高湿的贮藏环境中，随着时间的延长，鸡蛋的失重率在升高、哈氏单位和蛋黄指数在降低。H-M和SSPS涂膜组的鸡蛋品质显著优于其它组（L-M组、对照组和空白组）（P＜0.05），在第30d时这两组鸡蛋的品质仍为A级，而其它组均降为C级。从扫描电镜(SEM)对蛋壳结构的观察可知，SSPS和H-M对蛋壳上的气孔具有很好的封闭效果。
（3）分别采用磷酸酯化法和纳米SiOx法对SSPS进行化学改性得到改性产物SSPS-P和SSPS-S。分别以SSPS、SSPS-P和SSPS-S为主要原料，添加增稠剂海藻酸钠、辅助抑菌剂山梨酸钾配制成不同的鸡蛋涂膜保鲜剂，在室温条件下进行鸡蛋涂膜保鲜试验。SSPS、SSPS-P和SSPS-S对鸡蛋蛋黄指数的影响均无显著性差异（P＞0.05），而对鸡蛋失重率和哈氏单位的影响存在显著性差异（P＜0.05）。SSPS-S比SSPS和SSPS-P更能显著地减少鸡蛋失重率的上升、也更能显著地减弱鸡蛋哈氏单位的下降（P＜0.05）。
（4）对鸡蛋涂膜保鲜机理进行初步研究，主要通过扫描电镜、红外光谱、原子力显微镜、激光粒度分布仪以及高效液相色谱对水溶性大豆多糖及改性水溶性大豆多糖进行分析。得出其结构、性质有所不同。从而揭示对鸡蛋涂膜保鲜效果不同的原因。
Soluble Soybean Polysaccharide（SSPS）is a kind of water-soluble acid polysaccharide which isextracted from soybean cotyledon and has many excellent properties such as being high dietary fiber,stability against high fever, acid, salt, emulsification, low viscosity bacteriostasis and the ability offorming film. And its molecular weight ranges from5000to1000000. Based on the requirement forhealth and safety of food, SSPS can be used as the eggs coating material.
Previous study has been done for coating eggs with SSPS, but the effect was not very ideal.Because of the structural defects of polysaccharides, the bacteria restraint and water-resisting propertyof film forming with polysaccharides are very weak. Not only the usage amounts of SSPS and additivesare really big, but also the fresh-keeping result is not very good.
Therefore, the article studied the methods of physical and chemical modification of SSPS, chosethe right additives to prepare the eggs coating fresh-keeping solution, analyzed the fresh-keeping effectsusing the different coating materials and preliminarily probed into the fresh-keeping theory of SSPS andmodified SSPS. The specific research results are as follows:
(1) Rude SSPS was extracted from the wet soybean risidue by method of dilute acid hightemperature heat extraction, and its extraction ratio was34.2%. By comparing the effects of the ethanolprecipitation and ultrafiltration for separation and purification of rude SSPS, we found that the ratios ofthe extraction and the decolorization are31.4%,92.1%and33.9%,90.6%respectively. We got thedifferent molecular weight SSPS such as high molecular weight SSPS (H-M) and low molecular weightSSPS (L-M) by ultrafiltration which can concentrate the SSPS.
(2) Refined SSPS, H-M and L-M were employed as the main materials while sodium alginate wasemployed as thickener and potassium sorbet as bacteriostat to develop the fresh-keeping coating agents.The consistence（w/v） of main material, thickener and bacteriostat were3.0%,0.5%and0.6%respectively. During the high temperature and high humidity storage period, the Haugh unit and theYolk index of the SSPS and H-M coating eggs were significantly better than others(L-M, control andblank groups)（P＜0.05）. After30days storage, the quality level of the SSPS and H-M coating eggswas A grade, while the quality level of other groups dropped to C grade. From the structure of coatingeggshells which were observed through the scanning electron microscope (SEM), we knew that SSPSand H-M could sealed the pores of eggshells very well.
(3) SSPS were modified by two different chemical methods, one was phosphoric acid esterificationmethod and the other was nano SiOx method. We obtained the modified SSPS, noted for SSPS-P andSSPS-S respectively. Different eggs coating fresh-keeping solutions were prepared with SSPS, SSPS-Pand SSPS-S as main materials, sodium alginate as thickener and potassium sorbet as bacteriostat. Thecoatings were applied to shell eggs to examine potential effects on egg quality properties during30daysof storage at room temperature. The results indicated that all coatings can improve shelf-life of eggseffectively. Eggs index of eggs which were coated by SSPS、SSPS-P and SSPS-S had no significant difference（P＞0.05）. SSPS-S coating coated eggs showed higher Haugh unit and lower Weight loss
than the SSPS and SSPS-P coating coated eggs（P＜0.05）. But they were still better than the controlgroups.
(4) Scanning electron microscopy(SEM), Infrared Spectroscopy(IR), Atomic force microscope(AFM),the laser particle size distribution device and High performance liquid chromatography(HPLC) wereemployed to study the structures and properties of SSPS and modified SSPS, in order to explain thereason why they could prolong the shelf-life of eggs.
目前，已经有前人研究水溶性大豆多糖对鸡蛋的涂膜保鲜效果，但是效果不甚理想。这是由于多糖一些自身结构的限制，使得多糖制成的涂膜保鲜剂的抗菌性和抗水性较弱。而且前人研究的水溶性大豆多糖涂膜保鲜剂中水溶性大豆多糖和其它添加剂的使用量大，对鸡蛋的保鲜效果也不是特别明显。
因此，本试验主要研究水溶性大豆多糖的物理和化学改性，并且选择合适的添加剂配制成鸡蛋涂膜保鲜剂，对鸡蛋进行涂膜，研究其保鲜效果。同时，初步探究其对鸡蛋涂膜保鲜的机理。具体研究结果如下：
（1）采用弱酸高温热提取法从湿豆渣中提取粗制的水溶性大豆多糖，提取率为34.2%。然后分别用乙醇沉淀法和超滤法分离、纯化粗制的水溶性大豆多糖，其结果为：乙醇沉淀法的多糖得率和脱色率分别为31.4%和92.1%，超滤法的多糖得率和脱色率分别为33.9%和90.6%。通过超滤法还得到了不同相对分子质量的水溶性大豆多糖，并且超滤法还具有一定的浓缩效果。
（2）将水溶性大豆多糖经物理改性后得到高相对分子质量水溶性大豆多糖（H-M）和低相对分子质量水溶性大豆多糖（L-M）。分别以浓度为3.0%（w/v）的分离纯化的SSPS、H-M和L-M为主要原料，浓度为0.5%（w/v）的海藻酸钠作为增稠剂，浓度为0.6%（w/v）的山梨酸钾作为辅助抑菌剂，配制成不同的鸡蛋涂膜保鲜剂对鸡蛋进行涂膜保鲜试验。在高温、高湿的贮藏环境中，随着时间的延长，鸡蛋的失重率在升高、哈氏单位和蛋黄指数在降低。H-M和SSPS涂膜组的鸡蛋品质显著优于其它组（L-M组、对照组和空白组）（P＜0.05），在第30d时这两组鸡蛋的品质仍为A级，而其它组均降为C级。从扫描电镜(SEM)对蛋壳结构的观察可知，SSPS和H-M对蛋壳上的气孔具有很好的封闭效果。
（3）分别采用磷酸酯化法和纳米SiOx法对SSPS进行化学改性得到改性产物SSPS-P和SSPS-S。分别以SSPS、SSPS-P和SSPS-S为主要原料，添加增稠剂海藻酸钠、辅助抑菌剂山梨酸钾配制成不同的鸡蛋涂膜保鲜剂，在室温条件下进行鸡蛋涂膜保鲜试验。SSPS、SSPS-P和SSPS-S对鸡蛋蛋黄指数的影响均无显著性差异（P＞0.05），而对鸡蛋失重率和哈氏单位的影响存在显著性差异（P＜0.05）。SSPS-S比SSPS和SSPS-P更能显著地减少鸡蛋失重率的上升、也更能显著地减弱鸡蛋哈氏单位的下降（P＜0.05）。
（4）对鸡蛋涂膜保鲜机理进行初步研究，主要通过扫描电镜、红外光谱、原子力显微镜、激光粒度分布仪以及高效液相色谱对水溶性大豆多糖及改性水溶性大豆多糖进行分析。得出其结构、性质有所不同。从而揭示对鸡蛋涂膜保鲜效果不同的原因。
Soluble Soybean Polysaccharide（SSPS）is a kind of water-soluble acid polysaccharide which isextracted from soybean cotyledon and has many excellent properties such as being high dietary fiber,stability against high fever, acid, salt, emulsification, low viscosity bacteriostasis and the ability offorming film. And its molecular weight ranges from5000to1000000. Based on the requirement forhealth and safety of food, SSPS can be used as the eggs coating material.
Previous study has been done for coating eggs with SSPS, but the effect was not very ideal.Because of the structural defects of polysaccharides, the bacteria restraint and water-resisting propertyof film forming with polysaccharides are very weak. Not only the usage amounts of SSPS and additivesare really big, but also the fresh-keeping result is not very good.
Therefore, the article studied the methods of physical and chemical modification of SSPS, chosethe right additives to prepare the eggs coating fresh-keeping solution, analyzed the fresh-keeping effectsusing the different coating materials and preliminarily probed into the fresh-keeping theory of SSPS andmodified SSPS. The specific research results are as follows:
(1) Rude SSPS was extracted from the wet soybean risidue by method of dilute acid hightemperature heat extraction, and its extraction ratio was34.2%. By comparing the effects of the ethanolprecipitation and ultrafiltration for separation and purification of rude SSPS, we found that the ratios ofthe extraction and the decolorization are31.4%,92.1%and33.9%,90.6%respectively. We got thedifferent molecular weight SSPS such as high molecular weight SSPS (H-M) and low molecular weightSSPS (L-M) by ultrafiltration which can concentrate the SSPS.
(2) Refined SSPS, H-M and L-M were employed as the main materials while sodium alginate wasemployed as thickener and potassium sorbet as bacteriostat to develop the fresh-keeping coating agents.The consistence（w/v） of main material, thickener and bacteriostat were3.0%,0.5%and0.6%respectively. During the high temperature and high humidity storage period, the Haugh unit and theYolk index of the SSPS and H-M coating eggs were significantly better than others(L-M, control andblank groups)（P＜0.05）. After30days storage, the quality level of the SSPS and H-M coating eggswas A grade, while the quality level of other groups dropped to C grade. From the structure of coatingeggshells which were observed through the scanning electron microscope (SEM), we knew that SSPSand H-M could sealed the pores of eggshells very well.
(3) SSPS were modified by two different chemical methods, one was phosphoric acid esterificationmethod and the other was nano SiOx method. We obtained the modified SSPS, noted for SSPS-P andSSPS-S respectively. Different eggs coating fresh-keeping solutions were prepared with SSPS, SSPS-Pand SSPS-S as main materials, sodium alginate as thickener and potassium sorbet as bacteriostat. Thecoatings were applied to shell eggs to examine potential effects on egg quality properties during30daysof storage at room temperature. The results indicated that all coatings can improve shelf-life of eggseffectively. Eggs index of eggs which were coated by SSPS、SSPS-P and SSPS-S had no significant difference（P＞0.05）. SSPS-S coating coated eggs showed higher Haugh unit and lower Weight loss
than the SSPS and SSPS-P coating coated eggs（P＜0.05）. But they were still better than the controlgroups.
(4) Scanning electron microscopy(SEM), Infrared Spectroscopy(IR), Atomic force microscope(AFM),the laser particle size distribution device and High performance liquid chromatography(HPLC) wereemployed to study the structures and properties of SSPS and modified SSPS, in order to explain thereason why they could prolong the shelf-life of eggs.
引文
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44. Anker M. Mechanical properties water vapor permeability and moisture contents ofβ-Lactoglogulin and when protein films using multivariate analysis. Agric Food Chem.1998,(46):1820～1829.
45. Bhale S, No H K, Prinyawiwatkul W, et al. Chitosan coating improves shelf life of eggs[J]. FoodScience,2003,68(7):2378～2383.
46. Campaniello D, Beyilacqua A, Sinigaglia M, et al. Chitosan: antimicrobial activity and potentialapplications for preserving minimally processed st rawberries[J]. Food Microbiology,2008,25(8):9922～10000.
47. Durango A M, Soares N F F, Andrade, et al. Microbiological evaluation of an edible antimicrobialcoating on minimally processed carrots[J]. Food Control,2006,17(5),336～341.
48. Edyel A, Clarke M A, To N T. Near Infrared Analysis of Polysaccharides in sugar products.Abstracts of Papers American Chemical Society,1992,204:23~28.
49. Favier G. I., Escudero, M. E. Guzman, et al. Inhibitory effects of sucrose fatty acid esters onsurvival of Yersinia enterocolitica on eggshell surface and use of blue lake as indicator of bacterialpenetration into eggs．Journal of applied microbiology,2009,106(3):774~783．
50. Funk E.M.. The relation of the yolk index determined in natural position to the yolk index, asdetermined after separating the yolk form albumen. Poultry science,1948(27):367.
51. Geresh，S.， Yarmolinsky，I. A. E. et al. Characterization of the extracellular polysaccharide ofPorphyridium sp.： molecular weight determination and rheological properties. Carbohydr.Polym.，2002，50，183-189.
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57. Liu X.C. and Dordick J. S. Sugar-containing polyamines prepared using galactose oxidase coupledwith chemical reduction. J. Am. Chem. Soc.，121，466-467.
58. Mark A M, Roth W B, Mehltretter C L, et al. Oxygen permeability of amylomaize starch films[J].Food Technology,1996,20:75～80.
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60. Nakamura A, Takahashi T, Yoshida R, et al. Correding M. Mulsifying properties of soybean solublepolysaccharide. Food Hydrocolloids，2004，18：795-803.
61. Nakamura A, Furuta H, Maeda H et al. Structural studies by stepwise enzymatic degradation of themain backbone of soybean soluble polysaccharides consisting of galacturonan. Bioscience.Biotechnology. Biochemistry.2002,66(5):155~1158.
62. Nakamura A, Furuta H, Maeda H, et al. Analysis of structural components and molecularconstruction of soybean soluble polysaccharides by stepwise enzymatic degradation. Bioscience.Biotechnology. Biochemistry.2001,65(10):2249～2258.
63. Nakamura A, Furuta H, Maeda H, et al. Structural studies by stepwise enzymatic degradation of themain backbone of soybean soluble polysaccharides consisting of galacturonan. Bioscience.Biotechnology. Biochemistry.2002,66(5):1155～1158.
64. Nakamura A, Takahashi T, Yoshida R, et al. Mulsifying properties of soybean solublepolysaccharide. Food Hydrocolloids.2004,18:795～803.
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68. Rong M Z, Zhang M Q, Zeng H M, et al. St ructure-property relationships of irradiation graf tednano2inorganic particle filled polypropylene composites. Polymer,2001,42:167.
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75. Zak A., Obanu A. A., Mpieri．Efficiency of dietary vegetable oils preserving the quality of shelleggs under ambient tropical conditions．Journal of the Science of Food and Agriculture,2006,35:1311~1317
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43. Ana C. C. A, Aloiso J. A.. Internal Quality of Eggs Coated with Whey Protein Concentrate. Scienceand Agriculture,2004,61:276～280.
44. Anker M. Mechanical properties water vapor permeability and moisture contents ofβ-Lactoglogulin and when protein films using multivariate analysis. Agric Food Chem.1998,(46):1820～1829.
45. Bhale S, No H K, Prinyawiwatkul W, et al. Chitosan coating improves shelf life of eggs[J]. FoodScience,2003,68(7):2378～2383.
46. Campaniello D, Beyilacqua A, Sinigaglia M, et al. Chitosan: antimicrobial activity and potentialapplications for preserving minimally processed st rawberries[J]. Food Microbiology,2008,25(8):9922～10000.
47. Durango A M, Soares N F F, Andrade, et al. Microbiological evaluation of an edible antimicrobialcoating on minimally processed carrots[J]. Food Control,2006,17(5),336～341.
48. Edyel A, Clarke M A, To N T. Near Infrared Analysis of Polysaccharides in sugar products.Abstracts of Papers American Chemical Society,1992,204:23~28.
49. Favier G. I., Escudero, M. E. Guzman, et al. Inhibitory effects of sucrose fatty acid esters onsurvival of Yersinia enterocolitica on eggshell surface and use of blue lake as indicator of bacterialpenetration into eggs．Journal of applied microbiology,2009,106(3):774~783．
50. Funk E.M.. The relation of the yolk index determined in natural position to the yolk index, asdetermined after separating the yolk form albumen. Poultry science,1948(27):367.
51. Geresh，S.， Yarmolinsky，I. A. E. et al. Characterization of the extracellular polysaccharide ofPorphyridium sp.： molecular weight determination and rheological properties. Carbohydr.Polym.，2002，50，183-189.
52. Jones D. R.,Curtis P. A.,Anderson K. E., et a1．Microbial contami nation in inoculated shell eggs:II．Effects of layer strain and egg storage．Poultry Science,2004,83(1):95~100．
53. Kim S H, No H K, Prinyawiwatkul W, et al. Type of chitosan and chitosan solution pH on theshelf-life and quality of coated eggs[J]. Journal of Food Science,2007,72(1):44～48.
54. Kim S Y, No Hong Kyoon, Prinyawiwatkul. Effect of Molecular Weight, Type of Chitosan, andChitosan Solution pH on the Shelf-Life and Quality of Coated Eggs. Journal of Food Science,2007,7(1):44～48.
55. Kulicke W.M.，Iettau A. L.，Thielking H. Correlation between immunological activity， molarmass， and molecular structure of different (1→3) β-D-glucans. Carbohydr： Res.，1997，297：135-143.
56. Lin B F, Du Y M, Liang X Q. Effect of chitosan coating on respiratory behavior and quality ofstored litchi under ambient temperature[J]. Journal of Food Engineering,2011,102(1):94～99.
57. Liu X.C. and Dordick J. S. Sugar-containing polyamines prepared using galactose oxidase coupledwith chemical reduction. J. Am. Chem. Soc.，121，466-467.
58. Mark A M, Roth W B, Mehltretter C L, et al. Oxygen permeability of amylomaize starch films[J].Food Technology,1996,20:75～80.
59. Nakamura A, Furuta H., Maeda H., et al. Structural studies by stepwise enzymatic degradation ofthe main backbone of soybean soluble polysaccharides consisting of galacturonan. Bioscience.Biotechnology Biochemistry,2002,66(5),1155~1158.
60. Nakamura A, Takahashi T, Yoshida R, et al. Correding M. Mulsifying properties of soybean solublepolysaccharide. Food Hydrocolloids，2004，18：795-803.
61. Nakamura A, Furuta H, Maeda H et al. Structural studies by stepwise enzymatic degradation of themain backbone of soybean soluble polysaccharides consisting of galacturonan. Bioscience.Biotechnology. Biochemistry.2002,66(5):155~1158.
62. Nakamura A, Furuta H, Maeda H, et al. Analysis of structural components and molecularconstruction of soybean soluble polysaccharides by stepwise enzymatic degradation. Bioscience.Biotechnology. Biochemistry.2001,65(10):2249～2258.
63. Nakamura A, Furuta H, Maeda H, et al. Structural studies by stepwise enzymatic degradation of themain backbone of soybean soluble polysaccharides consisting of galacturonan. Bioscience.Biotechnology. Biochemistry.2002,66(5):1155～1158.
64. Nakamura A, Takahashi T, Yoshida R, et al. Mulsifying properties of soybean solublepolysaccharide. Food Hydrocolloids.2004,18:795～803.
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66. Padron M N. Salmonella typhimurium penetration through the eggshells of hatching eggs. AvianDisease,1990,34:463～468.
67. Rojas-grau M A, Soliva-fortuny R, Martin-belloso O. Edible coatings to incorporate activeingredients to freshcut fruits: a review[J]. Trends in Food Science and Technology,2009,20:438～
447.
68. Rong M Z, Zhang M Q, Zeng H M, et al. St ructure-property relationships of irradiation graf tednano2inorganic particle filled polypropylene composites. Polymer,2001,42:167.
69. Sanchez-gonzalez L, Pastir C, Vargas M, et al. Effect of hydroxypropylmethylcellulose andchitosan coatings with and without bergamot essential oil on quality and safety of cold-storedgrapes[J]. Postharvest Biology and Technology,2011,60(1):57～63.
70. Stadelman W J, Cotterill O J. Egg science and technology. New York: the Haworth Press Inc,1995.
71. Suppakul P, Jutakorn K, Bang Chokedee Y. Efficacy of cellulose-based coating on enhancing theshelf life of fresh eggs[J]. Unal of Food Engineering,2010,98(2):207～213.
72. Tabata K., Ito W., Kojima T. Ultrasonic degradation of schizophyllan, an antitumor polysaccharideproduced by schizophyllum commune fries. Carbohydr： Res.,1981,89：121-135.
73. Takahashi T, Maeda H, Aoyama T, et al. Physiological effects of water-soluble soybean fiber in rats.Biosci. Biotech Biochem.,1999,63(8):1340～1345.
74. Vangnai T, Wongs-aree C, Nimitkeatkal H, et al. Quality maintaining of ‘Daw’ longan usingchitosan coating[J]. Acta Horticulturae,2006,712(2):599～604.
75. Zak A., Obanu A. A., Mpieri．Efficiency of dietary vegetable oils preserving the quality of shelleggs under ambient tropical conditions．Journal of the Science of Food and Agriculture,2006,35:1311~1317