Present status on removal of raffinose family oligosaccharides – a Review J., Song G., Mei Y., Li R., Zhang H., Liu Y. (2019): Present status on removal of raff inose family oligosaccharides – a Review. Czech J. Food Sci., 37: 141-154.
download PDF

Raffinose family oligosaccharides (RFOs) are α-galactosyl derivatives of sucrose or glucose. They are found in a large variety of seeds from many different families such as beans, vegetables and whole grains. Due to absence of α-galactosidase in the digestive tract of humans and other monogastric animals, RFOs are responsible for intestinal disturbances (flatulence) following the ingestion of legume-derived products. Structural relationships of RFOs and their enzymatic degradation mechanism are described. Concentration and distribution from various seed sources are introduced. The present status on removal of the RFOs (such as soaking, cooking, germination, and addition of α-galactosidase) is summarized. At the meantime, α-galactosidases from botanic and microbial sources and their partial enzymatic properties are also presented in detail. Based on a comparison of various removal treatments, the microbial α-galactosidases are thought as the most optimum candidate for removing RFOs in legumes, and the ideal system for the RFO removal is proposed.

Abdel-Gawad A (1993): Effect of domestic processing on oligosaccharide content of some dry legume seeds. Food Chemistry, 46, 25-31
Alazzeh A.Y., Ibrahim S.A., Song D., Shahbazi A., AbuGhazaleh A.A. (2009): Carbohydrate and protein sources influence the induction of α- and β-galactosidases in Lactobacillus reuteri. Food Chemistry, 117, 654-659
Anisha G.S., Prema P. (2008): Reduction of non-digestible oligosaccharides in horse gram and green gram flours using crude α-galactosidase from Streptomyces griseoloalbus. Food Chemistry, 106, 1175-1179
Azeke Marshall A., Fretzdorff Barbara, Buening-Pfaue Hans, Betsche Thomas (2007): Comparative effect of boiling and solid substrate fermentation using the tempeh fungus (Rhizopus oligosporus) on the flatulence potential of African yambean (Sphenostylis stenocarpa L.) seeds. Food Chemistry, 103, 1420-1425
Backhed F. (2005): Host-Bacterial Mutualism in the Human Intestine. Science, 307, 1915-1920
Blöchl Andreas, Peterbauer Thomas, Richter Andreas (2007): Inhibition of raffinose oligosaccharide breakdown delays germination of pea seeds. Journal of Plant Physiology, 164, 1093-1096
BOREJSZO Z.BIGNIEW, KHAN K.HALIL (1992): Reduction of Flatulence-Causing Sugars by High Temperature Extrusion of Pinto Bean High Starch Fractions. Journal of Food Science, 57, 771-777
Brain J.C. (2013): Strategies for the removal of raffinose family oligosaccharides from navy bean flour. [Doctoral Thesis]. Palmerston North, Massey University.
Bulpin P.V., Gidley M.J., Jeffcoat R., Underwood D.R. (1990): Development of a biotechnological process for the modification of galactomannan polymers with plant α-galactosidase. Carbohydrate Polymers, 12, 155-168
Chen C. C., Shih Y. C., Chiou P. W. S., Yu B. (2010): Evaluating Nutritional Quality of Single Stage- and Two Stage-fermented Soybean Meal. Asian-Australasian Journal of Animal Sciences, 23, 598-606
Chi Chun-Hua, Cho Seong-Jun (2016): Improvement of bioactivity of soybean meal by solid-state fermentation with Bacillus amyloliquefaciens versus Lactobacillus spp. and Saccharomyces cerevisiae. LWT - Food Science and Technology, 68, 619-625
Chrost Bozena, Schmitz Klaus (2000): Purification and Characterization of Multiple Forms of α-Galactosidase in Cucumis melo Plants. Journal of Plant Physiology, 156, 483-491
Clarke J. H., Davidson K., Rixon J. E., Halstead J. R., Fransen M. P., Gilbert H. J., Hazlewood G. P. (2000): A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and α-galactosidase. Applied Microbiology and Biotechnology, 53, 661-667
CRUZ RUBEN, PARK YONG K. (1982): Production of Fungal ?-Galactosidase and Its Application to the Hydrolysis of Galactooligosaccharides in Soybean Milk. Journal of Food Science, 47, 1973-1975
DEFATIMAVIANA S, GUIMARAES V, JOSE I, DEALMEIDAEOLIVEIRA M, BRUNOROCOSTA N, DEBARROS E, MOREIRA M, DEREZENDE S (2005): Hydrolysis of oligosaccharides in soybean flour by soybean α-galactosidase. Food Chemistry, 93, 665-670
de Rezende S. T., Felix C. R. (1999): Production and characterization of raffinose-hydrolysing and invertase activities ofAspergillus fumigatus. Folia Microbiologica, 44, 191-195
Rezende Sebastião Tavares de, Guimarães Valéria Monteze, Rodrigues Marília de Castro, Felix Carlos Roberto (2005): Purification and characterization of an alpha-galactosidase from Aspergillus fumigatus. Brazilian Archives of Biology and Technology, 48, 195-202
Dey Prakash M., Naik Surbhi, Pridham John B. (1982): The lectin nature of α-galactosidases from Vicia faba seeds. FEBS Letters, 150, 233-237
DIBOFORI A, OKOH P, ONIGBINDE A (1994): Effect of germination on the cyanide and oligosaccharide content of lima beans (Phaseolus lunatus). Food Chemistry, 51, 133-136
Du Fang, Zhu Mengjuan, Wang Hexiang, Ng TziBin (2013): Purification and characterization of an α-galactosidase from Phaseolus coccineus seeds showing degrading capability on raffinose family oligosaccharides. Plant Physiology and Biochemistry, 69, 49-53
Egounlety M, Aworh O.C (2003): Effect of soaking, dehulling, cooking and fermentation with Rhizopus oligosporus on the oligosaccharides, trypsin inhibitor, phytic acid and tannins of soybean (Glycine max Merr.), cowpea (Vigna unguiculata L. Walp) and groundbean (Macrotyloma geocarpa Harms). Journal of Food Engineering, 56, 249-254
Falkoski Daniel L., Guimarães Valéria M., Callegari Carina M., Reis Angélica P., de Barros Everaldo G., de Rezende Sebastião T. (2006): Processing of Soybean Products by Semipurified Plant and Microbial α-Galactosidases. Journal of Agricultural and Food Chemistry, 54, 10184-10190
Fasina Oladiran, Tyler Bob, Pickard Mark, Zheng Guo-Hua, Wang Ning (2001): Effect of infrared heating on the properties of legume seeds *. International Journal of Food Science and Technology, 36, 79-90
Félix A.P., Rivera N.L.M., Sabchuk T.T., Lima D.C., Oliveira S.G., Maiorka A. (2013): The effect of soy oligosaccharide extraction on diet digestibility, faecal characteristics, and intestinal gas production in dogs. Animal Feed Science and Technology, 184, 86-93
Ferreira Joana Gasperazzo, Reis Angélica Pataro, Guimarães Valéria Monteze, Falkoski Daniel Luciano, da Silva Fialho Lílian, de Rezende Sebastião Tavares (2011): Purification and Characterization of Aspergillus terreus α-Galactosidases and Their Use for Hydrolysis of Soymilk Oligosaccharides. Applied Biochemistry and Biotechnology, 164, 1111-1125
Fialho Lílian da Silva, Guimarães Valéria Monteze, Callegari Carina Marin, Reis Angélica Pataro, Barbosa Daianny Silveira, Borges Eduardo Euclydes de Lima, Moreira Maurilio Alves, de Rezende Sebastião Tavares (2008): Characterization and biotechnological application of an acid α-galactosidase from Tachigali multijuga Benth. seeds. Phytochemistry, 69, 2579-2585
Ganter Christiane, Böck August, Buckel Peter, Mattes Ralf (1988): Production of thermostable, recombinant α-galactosidase suitable for raffinose elimination from sugar beet syrup. Journal of Biotechnology, 8, 301-310
Gao Zhifang, Schaffer Arthur A. (1999): A Novel Alkaline α-Galactosidase from Melon Fruit with a Substrate Preference for Raffinose. Plant Physiology, 119, 979-988
Garro Marisa S, de Valdez Graciela F, Oliver Guillermo, de Giori Graciela S (1996): Purification of α-galactosidase from Lactobacillus fermentum. Journal of Biotechnology, 45, 103-109
Girigowda K., Prashanth S. J., Mulimani V. H. (2005): Oligosaccharins of Black Gram (Vigna mungo L.) as Affected by Processing Methods. Plant Foods for Human Nutrition, 60, 173-180
Gote Manoj, Umalkar Harshali, Khan Islam, Khire Jayant (2004): Thermostable α-galactosidase from Bacillus stearothermophilus (NCIM 5146) and its application in the removal of flatulence causing factors from soymilk. Process Biochemistry, 39, 1723-1729
Gote M.M., Khan M.I., Gokhale D.V., Bastawde K.B., Khire J.M. (2006): Purification, characterization and substrate specificity of thermostable α-galactosidase from Bacillus stearothermophilus (NCIM-5146). Process Biochemistry, 41, 1311-1317
Grieshop Christine M., Kadzere Charles T., Clapper Gina M., Flickinger Elizabeth A., Bauer Laura L., Frazier Rodney L., Fahey George C. (2003): Chemical and Nutritional Characteristics of United States Soybeans and Soybean Meals. Journal of Agricultural and Food Chemistry, 51, 7684-7691
Guimarães Valéria Monteze, de Rezende Sebastião Tavares, Moreira Maurilio Alves, de Barros Everaldo Gonçalves, Felix Carlos Roberto (2001): Characterization of α-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides. Phytochemistry, 58, 67-73
Han In Hwa, Baik Byung-Kee (2006): Oligosaccharide Content and Composition of Legumes and Their Reduction by Soaking, Cooking, Ultrasound, and High Hydrostatic Pressure. Cereal Chemistry Journal, 83, 428-433
Hartwig Edgar E., Kuo Tsung Min, Kenty Michael M. (1997): Seed Protein and its Relationship to Soluble Sugars in Soybean. Crop Science, 37, 770-
Hymowitz T., Collins F. I. (1974): Variability of Sugar Content in Seed of Glycine max (L.) Merrill and G. soja Sieb. and Zucc.1. Agronomy Journal, 66, 239-
Iacono Giuseppe, Cavataio Francesca, Montalto Giuseppe, Florena Ada, Tumminello Mario, Soresi Maurizio, Notarbartolo Alberto, Carroccio Antonio (1998): Intolerance of Cow's Milk and Chronic Constipation in Children. New England Journal of Medicine, 339, 1100-1104
Ibrahim S.S., Habiba R.A., Shatta A.A., Embaby H.E. (2002): Effect of soaking, germination, cooking and fermentation on antinutritional factors in cowpeas. Molecular Nutrition & Food Research, 46: 92–95.
Kapnoor Shankar, Mulimani Veerappa Hanumanth (2010): Production of α-Galactosidase by Aspergillus oryzae through solid-state fermentation and its application in soymilk Galactooligosaccharide hydrolysis. Brazilian Archives of Biology and Technology, 53, 211-218
Karr-Lilienthal L.K., Kadzere C.T., Grieshop C.M., Fahey G.C. (2005): Chemical and nutritional properties of soybean carbohydrates as related to nonruminants: A review. Livestock Production Science, 97, 1-12
Katrolia Priti, Jia Huiyong, Yan Qiaojuan, Song Shuang, Jiang Zhengqiang, Xu Haibo (2012): Characterization of a protease-resistant α-galactosidase from the thermophilic fungus Rhizomucor miehei and its application in removal of raffinose family oligosaccharides. Bioresource Technology, 110, 578-586
Kotiguda G., Kapnoor S.S., Kulkarni D., Mulimani V.H. (2007): Degradation of raffinose oligosaccharides in soymilk by immobilized alpha-galactosidase of Aspergillus oryzae. Journal of Microbiology and Biotechnology, 17: 1430–1436.
Krishna Chundakkadu (2008): Solid-State Fermentation Systems—An Overview. Critical Reviews in Biotechnology, 25, 1-30
Lan Y., Williams B.A., Verstegen M.W.A., Patterson R., Tamminga S. (2007): Soy oligosaccharides in vitro fermentation characteristics and its effect on caecal microorganisms of young broiler chickens. Animal Feed Science and Technology, 133, 286-297
Linden James Carl (1982): Immobilized α-d-galactosidase in the sugar beet industry. Enzyme and Microbial Technology, 4, 130-136
Mansour Esam H, Khalil Ali H (1998): Reduction of raffinose oligosaccharides in chickpea (Cicer arietinum) flour by crude extracellular fungal α-galactosidase. Journal of the Science of Food and Agriculture, 78, 175-181<175::AID-JSFA100>3.0.CO;2-E
Manzanares Paloma, de Graaff Leo H, Visser Jaap (1998): Characterization of Galactosidases from Aspergillus niger: Purification of a Novel α-Galactosidase Activity. Enzyme and Microbial Technology, 22, 383-390
Matella N. J., Dolan K.D., Stoeckle A. W., Bennink M. R., Lee Y. S., Uebersax M. A. (2005): Use of Hydration, Germination, and α-Galactosidase Treatments to Reduce Oligosaccharides in Dry Beans. Journal of Food Science, 70, C203-C207
McCleary Barry V. (1983): Enzymic interactions in the hydrolysis of galactomannan in germinating guar: The role of exo-β-mannanase. Phytochemistry, 22, 649-658
Mulimani V.H., Ramalingam (1997): Enzymatic degradation of raffinose family sugars in chickpea flour. World Journal of Microbiology and Biotechnology, 13: 583–585.
Mulimani V.H., Thippeswamy S., Ramalingam S. (1997): Enzymatic degradation of oligosaccharides in soybean flours. Food Chemistry, 59, 279-282
Mulimani V.H, Devendra S (1998): Effect of soaking, cooking and crude α-galactosidase treatment on the oligosaccharide content of red gram flour. Food Chemistry, 61, 475-479
Naumova E.S., Korshunova I.V., Naumov G.I. (2003): Molecular analysis of the α-galactosidase MEL genes in yeast Saccharomyces sensu stricto. Molecular Biology, 37: 699–706.
Obelesu M., Bhagya S. (2006): Biochemical, functional and nutritional characteristics of soy protein concentrate prepared by thermal processing. Journal of Food Science and Technology, 43: 161–166.
Oboh H.A., Muzquiz M., Burbano C., Cuadrado C., Pedrosa M.M., Ayet G., Osagie A.U. (2000): Effect of soaking, cooking and germination on the oligosaccharide content of selected Nigerian legume seeds. Plant Foods for Human Nutrition, 55: 97–110.
Ozsoy Nurten, Berkkan Hakan (2003): Production and characterization of ?-galactosidase fromAspergillus flavipes. Cell Biochemistry and Function, 21, 387-389
Pandey Ashok, Soccol Carlos R, Nigam Poonam, Soccol Vanete T (2000): Biotechnological potential of agro-industrial residues. I: sugarcane bagasse. Bioresource Technology, 74, 69-80
Porter Jill E., Herrmann Klaus M., Ladisch Michael R. (1990): Integral kinetics of ?-galactosidase purified fromGlycine max for simultaneous hydrolysis of stachyose and raffinose. Biotechnology and Bioengineering, 35, 15-22
Prashanth S.J., Mulimani V.H. (2005): Soymilk oligosaccharide hydrolysis by Aspergillus oryzae α-galactosidase immobilized in calcium alginate. Process Biochemistry, 40, 1199-1205
Puchart V (): Purification and characterization of α-galactosidase from a thermophilic fungus Thermomyces lanuginosus. Biochimica et Biophysica Acta (BBA) - General Subjects, 1524, 27-37
Pugalenthi M., Siddhuraju P., Vadivel V. (2006): Effect of soaking followed by cooking and the addition of α-galactosidase on oligosaccharides levels in different Canavalia accessions. Journal of Food Composition and Analysis, 19, 512-517
Ramalingam, Panda R.P., Nachimuthu S., Sadasivam S. (2010): Degradation of flatulence-causing oligosaccharides in soymilk by α-galactosidase–A novel thermotolerant from Penicillium purpurogenum. Indian Journal of Biotechnology, 9: 160–165.
Rehms H., Barz W. (1995): Degradation of stachyose, raffinose, melibiose and sucrose by different tempe-producing Rhizopus fungi. Applied Microbiology and Biotechnology, 44, 47-52
Revilleza Ma. Jamela R., Mendoza Evelyn Mae T., Raymundo Leoncio C. (1990): Oligosaccharides in several Philippine indigenous food legumes: Determination, localization and removal. Plant Foods for Human Nutrition, 40, 83-93
Rezessy-Szabó J.M., Nguyen Q.D., Hoschke Á., Braet C., Hajós G., Claeyssens M. (2007): A novel thermostable α-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: Purification and characterization. Biochimica et Biophysica-General Subjects, 1770: 55–62.
Shadaksharaswamy M (): Changes in the oligosaccharides and the α-galactosidase content of coffee seeds during soaking and germination. Phytochemistry, 7, 715-719
SHANKAR S, MULIMANI V (2007): α-Galactosidase production by Aspergillus oryzae in solid-state fermentation. Bioresource Technology, 98, 958-961
Shankar S. K., Praveen Kumar S. K., Mulimani V. H. (2011): Calcium alginate entrapped preparation of α-galactosidase: its stability and application in hydrolysis of soymilk galactooligosaccharides. Journal of Industrial Microbiology & Biotechnology, 38, 1399-1405
Shen Wangyang, Jin Zhengyu, Xu Xueming, Zhao Jianwei, Deng Li, Chen Hanqing, Yuan Chao, Li Dandan, Li Xuehong (2008): New source of α-d-galactosidase: Germinating coffee beans. Food Chemistry, 110, 962-966
Shibuya Hajime, Kobayashi Hideyuki, Gun Park Gwi, Komatsu Yoko, Sato Taku, Kaneko Reiji, Nagasaki Hiroaki, Yoshida Shigeki, Kasamo Kunihiro, Kusakabe Isao (2014): Purification and Some Properties of α -Galactosidase from Penicillium purpurogenum. Bioscience, Biotechnology, and Biochemistry, 59, 2333-2335
Shibuya Hajime, Kobayashi Hideyuki, Sato Taku, Kim Wong-Sin, Yoshida Shigeki, Kaneko Satoshi, Kasamo Kunihiro, Kusakabe Isao (2014): Purification, Characterization, and cDNA Cloning of a Novel α -Galactosidase from Mortievella vinacea. Bioscience, Biotechnology, and Biochemistry, 61, 592-598
Shimelis Emire Admassu, Rakshit Sudip Kumar (2007): Effect of processing on antinutrients and in vitro protein digestibility of kidney bean (Phaseolus vulgaris L.) varieties grown in East Africa. Food Chemistry, 103, 161-172
Shiroya Tsugio (1963): Metabolism of raffinose in cotton seeds. Phytochemistry, 2, 33-46
Shivam Kumar, Mishra Sarad Kumar (2010): Purification and characterization of a thermostable α-galactosidase with transglycosylation activity from Aspergillus parasiticus MTCC-2796. Process Biochemistry, 45, 1088-1093
Simila J., Gernig A., Murray P., Fernandes S., Tuohy M.G. (2010): Cloning and expression of a thermostable α-galactosidase from the thermophilic fungus Talaromyces emersonii in the methylotrophic yeast Pichia pastoris. Journal of Microbiology and Biotechnology, 20: 1653–1663.
Singh U. (1988): Antinutritional factors of chickpea and pigeonpea and their removal by processing. Plant Foods for Human Nutrition, 38, 251-261
Singh Neelesh, Kayastha Arvind M. (2013): A novel application of Cicer α-galactosidase in reduction of raffinose family oligosaccharides in soybean flour. Journal of Plant Biochemistry and Biotechnology, 22, 353-356
Sinitsyna O. A., Fedorova E. A., Vakar I. M., Kondratieva E. G., Rozhkova A. M., Sokolova L. M., Bubnova T. M., Okunev O. N., Chulkin A. M., Vinetsky Y. P., Sinitsyn A. P. (2008): Isolation and characterization of extracellular α-galactosidases from Penicillium canescens. Biochemistry (Moscow), 73, 97-106
Somiari R. I., Balogh E. (1992): Hydrolysis of raffinose and stachyose in cowpea (Vigna unguiculata) flour, using ?-galactosidase fromAspergillus niger. World Journal of Microbiology & Biotechnology, 8, 564-566
Somiari Richard I, Balogh Esther (1993): Effect of soaking, cooking and crude α-galactosidase treatment on the oligosaccharide content of cowpea flours. Journal of the Science of Food and Agriculture, 61, 339-343
Somiari Richard I., Balogh Esther (1995): Properties of an extracellular glycosidase of Aspergillus niger suitable for removal of oligosaccharides from cowpea meal. Enzyme and Microbial Technology, 17, 311-316
Song Danfeng, Chang Sam K. C. (2006): Enzymatic Degradation of Oligosaccharides in Pinto Bean Flour. Journal of Agricultural and Food Chemistry, 54, 1296-1301
Subramaniyam R., Vimada R. (2012): Solid state and submerged fermentation for the production bioactive substances: A comparative study. International Journal of Science and Nature, 3: 480–486.
Thippeswamy S, Mulimani V.H (2002): Enzymic degradation of raffinose family oligosaccharides in soymilk by immobilized α-galactosidase from Gibberella fujikuroi. Process Biochemistry, 38, 635-640
Trugo L.C., Ramos L.A., Trugo N.M.F., Souza M.C.P. (1990): Oligosaccharide composition and trypsin inhibitor activity of P. vulgaris and the effect of germination on the α-galactoside composition and fermentation in the human colon. Food Chemistry, 36, 53-61
Tudor K.W., Jones M.A., Hughes S.R., Holt J.P., Wiegand B.R. (2013): Effect of fermentation with Saccharomyces cerevisiae strain PJ69-4 on the phytic acid, raffinose, and stachyose contents of soybean meal. The Professional Animal Scientist, 29, 529-534
Vijayakumari K., Siddhuraju P., Janardhanan K. (1996): Effect of soaking, cooking and autoclaving on phytic acid and oligosaccharide contents of the tribal pulse, Mucuna monosperma DC. ex. Wight. Food Chemistry, 55, 173-177
Wang Huimin, Ma Rui, Shi Pengjun, Huang Huoqing, Yang Peilong, Wang Yaru, Fan Yunliu, Yao Bin (2015): Insights into the substrate specificity and synergy with mannanase of family 27 α-galactosidases from Neosartorya fischeri P1. Applied Microbiology and Biotechnology, 99, 1261-1272
Wang Caihong, Wang Huimin, Ma Rui, Shi Pengjun, Niu Canfang, Luo Huiying, Yang Peilong, Yao Bin (2016): Biochemical characterization of a novel thermophilic α-galactosidase from Talaromyces leycettanus JCM12802 with significant transglycosylation activity. Journal of Bioscience and Bioengineering, 121, 7-12
Yamaguishi Caroline T., Sanada Cassia T., Gouvêa Patricia M., Pandey Ashok, Woiciechowski Adenise L., Parada José L., Soccol Carlos R. (2009): Biotechnological process for producing black bean slurry without stachyose. Food Research International, 42, 425-429
Yang Hailong, Zhang Liang, Xiao Gongnian, Feng Jiabin, Zhou Huabin, Huang Furu (2015): Changes in some nutritional components of soymilk during fermentation by the culinary and medicinal mushroom Grifola frondosa. LWT - Food Science and Technology, 62, 468-473
YOON M, HWANG H (2008): Reduction of soybean oligosaccharides and properties of α-d-galactosidase from Lactobacillus curvatus R08 and Leuconostoc mesenteriodes JK55. Food Microbiology, 25, 815-823
Zhang Jian, Zhang Shunxi, Yang Xinni, Qiu Ling, Gao Bing, Li Rui, Chen Jiwang (2016): Reactive extraction of amino acids mixture in hydrolysate from cottonseed meal with di(2-ethylhexyl) phosphoric acid. Journal of Chemical Technology & Biotechnology, 91, 483-489
download PDF

© 2019 Czech Academy of Agricultural Sciences