foodsystFood systemsПищевые системы2618-97712618-7272Федеральный научный центр пищевых систем им. В.М. Горбатова РАН10.21323/2618-9771-2021-4-4-269-277foodsyst-135Research ArticleСтатьиUse of pullulanase as a biocatalyst for starch hydrolysis: Part 1. Study of the effect of pullulanase on maize amylopectin starchИспользование пуллуланазы в качестве биокатализатора процесса гидролиза крахмала. Часть 1. Изучение действия пуллуланазы на амилопектиновый кукурузный крахмалhttps://orcid.org/0000-0003-1178-8254ПапахинА. А.PapakhinA. A.

Папахин Александр Алексеевич — кандидат технических наук, заведующий лабораторией Биотехнологии крахмалопаточного сырья

140051, Московская обл., г.о Люберцы, д. п. Красково, ул. Некрасова, д. 11Тел.: +7–495–557–15–00

Alexander A. Papakhin, Сandidate of Technical Sciences, Head of the Laboratory of Biotechnology of starch raw materials

Nekrasov Str., 11, item Kraskovo, Lyubertsy district, Moscow regionTel.: +7–495–557–15–00

papahin_aleksandr@mail.ruhttps://orcid.org/0000-0001-9636-1537БородинаЗ. М.BorodinaZ. M.

Бородина Зинаида Михайловна — кандидат технических наук, ведущий научный сотрудник, лаборатория Биотехнологии крахмалопаточного сырья

140051, Московская обл., г.о Люберцы, д. п. Красково, ул. Некрасова, д. 11Тел.: +7–495–557–15–00

Zinaida M. Borodina, Candidate of Technical Sciences, Senior Researcher, Laboratory of Biotechnology of starch raw materials

Nekrasov Str., 11, item Kraskovo, Lyubertsy district, Moscow regionTel.: +7–495–557–15–00

borodina7292@yandex.ruВсероссийский научно-исследовательский институт крахмалопродуктовРоссияAll-Russian Scientific Research Institute for Starch ProductsRussian Federation20210501202244269277Copyright © Papakhin A.A., Borodina Z.M., 20222022Папахин А.А., Бородина З.М.Papakhin A.A., Borodina Z.M.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.fsjour.com/jour/article/view/135

The use of debranching enzymes in starch hydrolysis is a topical direction for obtaining new types of starch products with controlled properties and a potential for the further use. The aim of the work was to study an effect of pullulanase (EC3.2.1.41) on maize amylopectin starch in the native and gelatinized state. The objects of the research were maize amylopectin starch and enzyme preparation Promozyme D6 (Novozymes, Denmark). High-performance liquid chromatography (HPLC) was used to determine the carbohydrate composition of hydrolysates. The mass fraction of reducing substances (RS) was determined by the Lane and Eynon method. A rotational viscometer was used to measure dynamic viscosity of the starch hydrolysis products. It was found that analyzed starch in the native state showed low enzymatic sensitivity to the action of pullulanase with insignificant changes in viscosity, solubility and iodine binding capacity of the samples. Pullulanase showed the highest effect on gelatinized starch during the first eight hours of incubation. After eight hours, the maximum degree of starch hydrolysis by pullulanase at a dose of 10 units/g dry matter (DM) was 4.7% on DM basis, iodine binding capacity of the hydrolysate was D600 = 0.343 (in the control experiment D600 = 0.154), and the viscosity of the hydrolysate decreased from 7887 mPa · s to 4.3 mPa · s. Hydrolysates cooled to 8 °C and held for 20 hours along with hydrolysates that were not cooled showed high susceptibility to attack by glucoamilase (97–98%) at 60 °C and 24 hours of saccharification, which suggested the absence of their resistance to the action of glucoamilase in the conditions of the experiment. The use of pullulanase in dextrinization of the analyzed starch, which was gelatinized and partly hydrolyzed by α-amylase (RS6.1%), enabled obtaining hydrolysates with the mass fraction of reducing substances in a range of 10–24% on DM basis with the process duration of 2 to 24 hours and the enzyme dose of 2–10 units, which contained mainly maltotriose, maltohexose and maltoheptose with their total amount of 45–60% on DM basis. The results indicate a need for further research of the biocatalytic action of pullulanase to develop new methods for enzymatic modification of starch.

Использование деразветвляющих ферментов при гидролизе крахмала является актуальным направлением для получения новых видов крахмалопродуктов с контролируемыми свойствами и потенциалом для дальнейшего использования. Целью работы являлось изучение действия пуллуланазы (ЕС 3.2.1.41) на кукурузный амилопектиновый крахмал в нативном и клейстеризованном состоянии. Объектами исследований являлись амилопектиновый кукурузный крахмал и ферментный препарат Promozyme D6 (Novozymes, Дания). Для определения углеводного состава гидролизатов применяли метод высокоэффективной жидкостной хроматографии (ВЭЖХ), массовую долю редуцирующих веществ (РВ) определяли методом Лейна и Эйнона, для измерения динамической вязкости продуктов гидролиза крахмала был использован ротационный вискозиметр. Выявлено, что в нативном состоянии испытуемый крахмал проявил невысокую ферментативную восприимчивость к действию пуллуланазы с незначительными изменениями вязкости, растворимости и йодсвязующей способности образцов. Показано, что наибольшую активность на клейстеризованный крахмал пуллуланаза проявляла в первые 8 часов инкубации. Установлено, что максимальная степень гидролиза крахмала пуллуланазой через 8 часов при дозе 10 ед/г сухого вещества (СВ) составила 4,7% по СВ, йодсвязующая способность гидролизата D600 –0,343, при этом в контрольном опыте она составила D600 –0,154, а вязкость гидролизата снизилась с 7887 мПас · с до 4,3 мПа · с. Гидролизаты, охлажденные до 8 °C и выдержанные в течение 20 часов наряду с неохлажденными, проявили высокую атакуемость глюкоамилазой на 97–98% при 60 °C и 24 часа осахаривания, что указывало на отсутствие их резистентности к действию глюкоамилазы в условиях опыта. Использование пуллуланазы при декстринизации клейстеризованного и частично гидролизованного α-амилазой (РВ 6,1%) испытуемого крахмала позволяло получать гидролизаты с массовой долей редуцирующих веществ в пределах 10–24% по СВ при продолжительности процесса от 2 до 24 часов и дозировке фермента 2–10 ед., которые содержали в основном мальтотриозу, мальтогексозу и мальтогептозу с их суммарным количеством 45–60% по СВ. Результаты свидетельствуют о необходимости продолжения исследований биокаталитического действия пуллуланазы для разработки новых способов ферментативной модификации крахмала.

амилолитические ферментыамилозаамилопектинструктурные свойстваредуцирующие веществауглеводный составamylolytic enzymesamyloseamylopectinstructural propertiesreducing substancescarbohydrate compositionСтатья подготовлена в рамках выполнения исследований по государственному заданию № 05 Федерального научного центра пищевых систем им. В. М. Горбатова Российской академии наук.The article was published as part of the research topic No. 0585–2019–033-С-01 of the state assignment of the V. M. Gorbatov Federal Research Center for Food Systems of RAS.ReferencesRaveendran, S., Parameswaran B., Ummalyma, S. B., Abraham, A., Mathew, A.K., Madhavan, A. et al. (2018). Applications of microbial enzymes in food industry. Food Technology and Biotechnology, 56(1), 16–30. https://doi.org/10.17113/ftb.56.01.18.5491Raveendran, S., Parameswaran B., Ummalyma, S. B., Abraham, A., Mathew, A.K., Madhavan, A. et al. (2018). Applications of microbial enzymes in food industry. Food Technology and Biotechnology, 56(1), 16–30. https://doi.org/10.17113/ftb.56.01.18.5491Shinde, V., Deshmukh, S., Bhoyar, M.G. (2015). Applications of major enzymes in food industry. Indian Farmer, 2(6), 497–502.Shinde, V., Deshmukh, S., Bhoyar, M.G. (2015). Applications of major enzymes in food industry. Indian Farmer, 2(6), 497–502.Hashim, S.O. (2020). Starch-modifying enzymes. Advances in Biochemical Engineering/Biotechnology,172, 221–244. https://doi.org/10.1007/10_2019_91Hashim, S.O. (2020). Starch-modifying enzymes. Advances in Biochemical Engineering/Biotechnology,172, 221–244. https://doi.org/10.1007/10_2019_91Tester, R.F., Karkalas J., Qi X. (2004). Starch structure and digestibility enzyme-substrate relationship. World’s Poultry Science Journal, 60(2), 186–195+248+250. https://doi.org/10.1079/WPS20040014Tester, R.F., Karkalas J., Qi X. (2004). Starch structure and digestibility enzyme-substrate relationship. World’s Poultry Science Journal, 60(2), 186–195+248+250. https://doi.org/10.1079/WPS20040014Klaochanpong, N., Puttanlek, C., Rungsardthong, V., Rungsardthong V., Puncha-arnon, S., Uttapap, D. (2015). Physicochemical and structural properties of debranched waxy rice, waxy corn and waxy potato starches. Food Hydrocolloids, 45, 218–226. https://doi.org/10.1016/j.foodhyd.2014.11.010Klaochanpong, N., Puttanlek, C., Rungsardthong, V., Rungsardthong V., Puncha-arnon, S., Uttapap, D. (2015). Physicochemical and structural properties of debranched waxy rice, waxy corn and waxy potato starches. Food Hydrocolloids, 45, 218–226. https://doi.org/10.1016/j.foodhyd.2014.11.010Hii, S.L., Tan, J.S., Ling, T.C., Ariff, A.B. (2012). Pullulanase: role in starch hydrolysis and potential industrial application. Enzyme research, 2012, Article 921362. https://doi.org/10.1155/2012/921362Hii, S.L., Tan, J.S., Ling, T.C., Ariff, A.B. (2012). Pullulanase: role in starch hydrolysis and potential industrial application. Enzyme research, 2012, Article 921362. https://doi.org/10.1155/2012/921362Manners, D.J. (1997). Observations of the specifity and nomenclature of starch debranching enzymes. Journal of Applied Glycoscience, 44, 83–85.Manners, D.J. (1997). Observations of the specifity and nomenclature of starch debranching enzymes. Journal of Applied Glycoscience, 44, 83–85.Xia, W., Zhang, K., Su, L., Wu, J. (2021). Microbial starch debranching enzymes: Developments and applications. Biotechnology Advances, 50, Article 107786. https://doi.org/10.1016/j.biotechadv.2021.107786Xia, W., Zhang, K., Su, L., Wu, J. (2021). Microbial starch debranching enzymes: Developments and applications. Biotechnology Advances, 50, Article 107786. https://doi.org/10.1016/j.biotechadv.2021.107786Xu, P., Zhang, S.-Y., Luo, Z.-G., Zong, M.-H., Li, X.-X., Lou, W.-Y. (2021). Biotechnology and bioengineering of pullulanase: state of the art and perspectives. World Journal of Microbiology and Biotechnology, 37(3), Article 43. https://doi.org/10.1007/s11274–021–03010–9Xu, P., Zhang, S.-Y., Luo, Z.-G., Zong, M.-H., Li, X.-X., Lou, W.-Y. (2021). Biotechnology and bioengineering of pullulanase: state of the art and perspectives. World Journal of Microbiology and Biotechnology, 37(3), Article 43. https://doi.org/10.1007/s11274–021–03010–9Соловьева, С.Ю. (2004). Разработка технологии биоконверсии крахмала при производстве патоки различного углеводного состава. Автореф. дис. канд. техн. наук. Москва, МГУПП. — 25 с.Solovyova, S. Yu. (2004). Development of starch bioconversion technology in the production of molasses with various carbohydrate composition. Author’s abstract of the dissertation for the scientific degree of Candidate of Technical Sciences. Moscow, MGUPP, 25 p. (In Russian)Li, C., Kong, H., Yang, Q., Gu, Z., Ban, X., Cheng, L. et al. (2020). A temperature-mediated two-step saccharification process enhances maltose yield from high-concentration maltodextrin solutions. Journal of the Science of Food and Agriculture, 101(9), 3742–3748. https://doi.org/10.1002/jsfa.11005Li, C., Kong, H., Yang, Q., Gu, Z., Ban, X., Cheng, L. et al. (2020). A temperature-mediated two-step saccharification process enhances maltose yield from high-concentration maltodextrin solutions. Journal of the Science of Food and Agriculture, 101(9), 3742–3748. https://doi.org/10.1002/jsfa.11005Xie, A.-J., Lee, D.-J., Lim, S.-T. (2021). Characterization of resistant waxy maize dextrins prepared by simultaneous debranching and crystallization followed by acidic or enzymatic hydrolysis. Food Hydrocolloids, 121, Aticle 106942. https://doi.org/10.1016/j.foodhyd.2021.106942Xie, A.-J., Lee, D.-J., Lim, S.-T. (2021). Characterization of resistant waxy maize dextrins prepared by simultaneous debranching and crystallization followed by acidic or enzymatic hydrolysis. Food Hydrocolloids, 121, Aticle 106942. https://doi.org/10.1016/j.foodhyd.2021.106942Arp, C. G., Correa, M. J., Ferrero, C. (2020). Production and characterization of type III resistant starch from native wheat starch using thermal and enzymatic modifications. Food and Bioprocess Technology, 13(7), 1181–1192. https://doi.org/10.1007/s11947–020–02470–5Arp, C. G., Correa, M. J., Ferrero, C. (2020). Production and characterization of type III resistant starch from native wheat starch using thermal and enzymatic modifications. Food and Bioprocess Technology, 13(7), 1181–1192. https://doi.org/10.1007/s11947–020–02470–5Li, H., Li, J., Xiao, Y., Cui, B., Fang, Y., Guo, L. (2019). In vitro digestibility of rice starch granules modified by β-amylase, transglucosidase and pullulanase. International Journal of Biological Macromolecules, 136, 1228-1236. https://doi.org/10.1016/j.ijbiomac.2019.06.111Li, H., Li, J., Xiao, Y., Cui, B., Fang, Y., Guo, L. (2019). In vitro digestibility of rice starch granules modified by β-amylase, transglucosidase and pullulanase. International Journal of Biological Macromolecules, 136, 1228-1236. https://doi.org/10.1016/j.ijbiomac.2019.06.111Li, P., He, X., Dhital, S., Zhang, B., Huang, Q. (2017). Structural and physicochemical properties of granular starches after treatment with debranching enzyme. Carbohydrate Polymers, 169, 351–356. https://doi.org/10.1016/j.carbpol.2017.04.036Li, P., He, X., Dhital, S., Zhang, B., Huang, Q. (2017). Structural and physicochemical properties of granular starches after treatment with debranching enzyme. Carbohydrate Polymers, 169, 351–356. https://doi.org/10.1016/j.carbpol.2017.04.036Jung, K.-H., Kim, M.-J., Park, S.-H., Hwang, H.-S., Lee, S., Shim, J.-H. et al. (2013). The effect of granule surface area on hydrolysis of native starches by pullulanase. Starch — Stärke, 65(9–10), 848–853. https://doi.org/10.1002/star.201200226Jung, K.-H., Kim, M.-J., Park, S.-H., Hwang, H.-S., Lee, S., Shim, J.-H. et al. (2013). The effect of granule surface area on hydrolysis of native starches by pullulanase. Starch — Stärke, 65(9–10), 848–853. https://doi.org/10.1002/star.201200226Hong, Y., Liu, G., Gu, Z. (2015). Preparation and characterization of hydrophilic debranched starch modified by pullulanase on swollen granule starch. Food Research International, 67, 212–218. https://doi.org/10.1016/j.foodres.2014.11.020Hong, Y., Liu, G., Gu, Z. (2015). Preparation and characterization of hydrophilic debranched starch modified by pullulanase on swollen granule starch. Food Research International, 67, 212–218. https://doi.org/10.1016/j.foodres.2014.11.020Xu, R, Xu, D.-H., Xu, H.-H. (2012). Effect of enzymatic hydrolysis on the resistant starch yield. Italian Journal of Food Science, 24(4), 367–375.Xu, R, Xu, D.-H., Xu, H.-H. (2012). Effect of enzymatic hydrolysis on the resistant starch yield. Italian Journal of Food Science, 24(4), 367–375.Gao, Q, Li, S., Jian, H., Liang, S. (2011). Preparation and properties of resistant starch from corn starch with enzymes. African Journal of Biotechnology, 10(7), 1186–1193.19.Gao, Q, Li, S., Jian, H., Liang, S. (2011). Preparation and properties of resistant starch from corn starch with enzymes. African Journal of Biotechnology, 10(7), 1186–1193.Shi, J., Sweedman, M. C., Shi, Y.-C. (2018). Structural changes and digestibility of waxy maize starch debranched by different levels of pullulanase. Carbohydrate Polymers, 194, 350–356. https://doi.org/10.1016/j.carbpol.2018.04.053Shi, J., Sweedman, M. C., Shi, Y.-C. (2018). Structural changes and digestibility of waxy maize starch debranched by different levels of pullulanase. Carbohydrate Polymers, 194, 350–356. https://doi.org/10.1016/j.carbpol.2018.04.053ГОСТ 32159–2013 «Крахмал кукурузный. Общие технические условия». — Москва: Стандартинформ, 2019. — 12 с.GOST 32159–2013 “Maize starch. General specifications”. — Moscow: Standartinform, 2019. —12 p. (In Russian)ГОСТ 34440–2018 «Ферментные препараты для пищевой промышленности. Методы определения амилолитической активности». Москва: Стандартинформ, 2018. —18 с.GOST 34440–2018 “Enzyme preparations for food industry. Methods for the determination of amylase activity”. — Moscow: Standartinform, 2018. — 18 p. (In Russian)ГОСТ 7698–93 «Крахмал. Правила приемки и методы анализа». Минск: Издательство стандартов, 2001. — 43 с.GOST 7698–93 “Starch. Acceptance rules and methods of analysis”. Minsk: Publishing House of Standards, 2001. —43 p. (In Russian)ГОСТ 33917–2016 «Патока крахмальная. Общие технические условия». — Москва: Стандартинформ, 2017. — 52 с.GOST 33917–2016 “Starch syrup. General specifications”. — Moscow: Standartinform, 2017. — 52 p. (In Russian)ГОСТ Р 50549–93 (ИСО 5377–81) «Продукты гидролиза крахмала. Определение восстанавливающей способности и эквивалента глюкозы. Метод постоянного титра Лейна и Эйнона». — Москва: Издательство стандартов, 1994. — 11 с.GOST R50549–93 (ISO 5377–81) “Starch hydrolysis products. Determination of reducing power and dextrose equiwalent Lane and Eynon constant titre method”. — Moscow: Publishing House of Standards, 1994. 11 p. (In Russian)ГОСТ ISO 6647–1–2015 «Рис. Определение содержания амилозы. Часть 1. Контрольный метод». — Москва: Стандартинформ, 2015. — 16 с.GOST ISO 6647–1–2015 “Rice. Determination of amylose content. Part 1. Reference method”. — Moscow: Standartinform, 2015. — 16 p. (In Russian)Лукин, Н.Д., Ананских, В.В., Лапидус, Т.В., Хворова, Л.С. (2007). Технологический контроль производства сахаристых крахмалопродуктов. Москва: Россельхозакадемия, 2007. — 261с.Lukin, N.D., Ananskikh, V.V., Lapidus, T.V., Khvorova, L.S. (2007). Technological control of the production of sugary starch products. Moscow: Russian Agricultural Academy. 2007, — 261p. (In Russian)Wasserman, L.A., Papachin, A.A., Borodina, Z.M., Krivandin, A.V., Sergeev, A.I., Tarasov, V.F. (2019). Some physico-chemical and thermodynamic characteristics of maize starches hydrolyzed by glucoamylase. Carbohydrate Polymers, 212, 260–269. https://doi.org/10.1016/j.carbpol.2019.01.096Wasserman, L.A., Papachin, A.A., Borodina, Z.M., Krivandin, A.V., Sergeev, A.I., Tarasov, V.F. (2019). Some physico-chemical and thermodynamic characteristics of maize starches hydrolyzed by glucoamylase. Carbohydrate Polymers, 212, 260–269. https://doi.org/10.1016/j.carbpol.2019.01.096Папахин, А.А., Бородина, З.М., Лукин, Н.Д., Гулакова, В.А., Маннова И. Г. (2014). Методика оценки действия оценки действия амилолитических ферментов на нативный крахмал. Хранение и переработка сельхозсырья, 4, 14–17.Papakhin, A.A., Borodina, Z.M., Lukin, N.D., Kulakova, V.A., Manova, I.G. (2014). Assessment methodology of amylolytic enzymes actions on native starch. Storage and Processing of Farm Products, 4, 14–17. (In Russian)Папахин, А.А, Бородина, З.М. (28–30 октября 2020 года). Действие глюкоамилазы на различные виды крахмала в нативном состоянии. Материалы международного конгресса «Биотехнология: состояние и перспективы развития». Москва: ООО «Экспобиохим-технологии», Россия, 2020. https://doi.org/10.37747/2312-640X-2020–18–284–285Papakhin, A.A., Borodina, Z.M. (28–30 Oktober, 2020). The effect of glucoamylase on various types of starch in the native state. Proceedings of international congress “Biotechnology: State of the art and perspectives”. Moscow: EXPO-Biohim-Technologies LLC, Russia, 2020. https://doi.org/10.37747/2312–640X-2020–18–284–285 (In Russian)Šárka, E., Dvořáček, V. (2017). Waxy starch as a perspective raw material (a review). Food Hydrocolloids, 69, 402–409. https://doi.org/10.1016/j.foodhyd.2017.03.001Šárka, E., Dvořáček, V. (2017). Waxy starch as a perspective raw material (a review). Food Hydrocolloids, 69, 402–409. https://doi.org/10.1016/j.foodhyd.2017.03.001Liu, W., Hong, Y., Gu, Z., Cheng, L., Li, Z., Li, C. (2017). In structure and in vitro digestibility of waxy corn starch debranched by pullulanase. Food Hydrocolloids, 67, 104–110. https://doi.org/10.1016/j.foodhyd.2016.12.036Liu, W., Hong, Y., Gu, Z., Cheng, L., Li, Z., Li, C. (2017). In structure and in — vitro digestibility of waxy corn starch debranched by pullulanase. Food Hydrocolloids, 67, 104–110. https://doi.org/10.1016/j.foodhyd.2016.12.036Shi, M., Chen, Y., Yu, S., Gao, Q. (2013). Preparation and properties of RS III from waxy maize starch with pullulanase. Food Hydrocolloids, 33(1), 19–25. https://doi.org/10.1016/j.foodhyd.2013.02.018Shi, M., Chen, Y., Yu, S., Gao, Q. (2013). Preparation and properties of RS III from waxy maize starch with pullulanase. Food Hydrocolloids, 33(1), 19–25. https://doi.org/10.1016/j.foodhyd.2013.02.018

The authors declare that there are no conflicts of interest present.