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Pullulanase M1 (Klebsiella planticola)

Product code: E-PULKP

700 Units

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Content: 700 Units
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Formulation: In 3.2 M ammonium sulphate
Physical Form: Suspension
Stability: > 4 years at 4oC
Enzyme Activity: Pullulanase/Limit-dextrinase
EC Number:
CAZy Family: GH13
CAS Number: 9075-68-7
Synonyms: pullulanase; pullulan 6-alpha-glucanohydrolase
Source: Klebsiella planticola
Molecular Weight: 109,000
Concentration: Supplied at ~ 650 U/mL
Expression: Purified from Klebsiella planticola
Specificity: Hydrolysis of (1,6)-α-D-glucosidic linkages in pullulan, amylopectin and glycogen, and in the α- and β-limit dextrins of amylopectin and glycogen.
Specific Activity: ~ 30 U/mg (40oC, pH 5.0 on pullulan)
Unit Definition: One Unit of pullulanase activity is defined as the amount of enzyme required to release one µmole of glucose reducing-sugar-equivalents per minute from pullulan (5 mg/mL) in sodium acetate buffer (100 mM), pH 5.0 at 40oC.
Temperature Optima: 40oC
pH Optima: 5
Application examples: Applications in the cereals, food and feeds industries particularly in starch saccharification and production of high glucose or maltose syrups.

High purity Pullulanase M1 (Klebsiella planticola) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Recommended pullulanase for research on starch structure.

View all Carbohydrate Active EnZyme products available.

Certificate of Analysis
Safety Data Sheet
FAQs Data Sheet

Slowly digestible property of highly branched α-limit dextrins produced by 4, 6-α-glucanotransferase from Streptococcus thermophilus evaluated in vitro and in vivo.

Ryu, J. J., Li, X., Lee, E. S., Li, D. & Lee, B. H. (2021). Carbohydrate Polymers, 275, 118685.

Starch molecules are first degraded to slowly digestible α-limit dextrins (α-LDx) and rapidly hydrolyzable linear malto-oligosaccharides (LMOs) by salivary and pancreatic α-amylases. In this study, we designed a slowly digestible highly branched α-LDx with maximized α-1,6 linkages using 4,6-α-glucanotransferase (4,6-αGT), which creates a short length of α-1,4 side chains with increasing branching points. The results showed that a short length of external chains mainly composed of 1–8 glucosyl units was newly synthesized in different amylose contents of corn starches, and the α-1,6 linkage ratio of branched α-LDx after the chromatographical purification was significantly increased from 4.6% to 22.1%. Both in vitro and in vivo studies confirmed that enzymatically modified α-LDx had improved slowly digestible properties and extended glycemic responses. Therefore, 4,6-αGT treatment enhanced the slowly digestible properties of highly branched α-LDx and promises usefulness as a functional ingredient to attenuate postprandial glucose homeostasis.

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Fine structure impacts highly concentrated starch liquefaction process and product performance.

Kong, H., Yu, L., Gu, Z., Li, Z., Ban, X., Cheng, L., Hong, Y. & Li, C. (2021). Industrial Crops and Products, 164, 113347.

Designing a highly concentrated (45 %, w/w) starch liquefaction process is a green method to enhance the productivity of starch syrup and related fermentation products. Previous studies mainly focused on handling highly concentrated normal corn starch slurries, but the production efficiency and product performance cannot perfectly match the conventional liquefaction process (30 %, w/w). In the present research, four starches from various botanical sources were selected with an objective to accelerate highly concentrated starch liquefaction process. The results demonstrated that with potato starch or tapioca starch as a substrate, liquefaction process was more feasible as observed from the obvious reduction in paste viscosity and acceleration in amylolysis. To clarify the mechanism of these differences, changes in the fine structure during liquefaction were further characterized. The long external chains (16.2 glucose units on average) in potato starch and long internal chains (5.1 glucose units on average) in tapioca starch, which indicated high proportion of consecutive α-1,4 linkages, seemed more susceptible to enzymatic attack under highly concentrated substrate condition. This caused rapid degradation of starch molecules. The liquefied products were suitable for glucose syrup production. By comparison, normal corn starch and waxy corn starch, which contain relatively shorter linear fragments, were less accessible to α-amylase. This suppressed liquefaction process led to the survival of large molecules, thereby being unsuitable for subsequent saccharification process. The results suggest that selecting an appropriate substrate is an effective strategy to accelerate highly concentrated starch liquefaction and improve product performance.

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Two 1, 4-α-glucan branching enzymes successively rearrange glycosidic bonds: A novel synergistic approach for reducing starch digestibility.

Yu, L., Kong, H., Gu, Z., Li, C., Ban, X., Cheng, L., Hong, Y. & Li, Z. (2021). Carbohydrate Polymers, 262, 117968.

Enzymatically rearranging α-1,4 and α-1,6 glycosidic bonds in starch is a green approach to regulating its digestibility. A two-step modification process successively catalyzed by 1,4-α-glucan branching enzymes (GBEs) from Rhodothermus obamensi STB05 (Ro-GBE) and Geobacillus thermoglucosidans STB02 (Gt-GBE) was investigated as a strategy to reduce the digestibility of corn starch. This dual GBE modification process caused a reduction of 25.8 % in rapidly digestible starch fraction in corn starch, which were more effective than single GBE-catalyzed modification with the same duration. Structural analysis indicated that the dual GBE modified product contained higher branching density, more abundant short branches, and shorter external chains than those in single GBE-modified product. These results demonstrated that a moderate Ro-GBE treatment prior to starch gelatinization caused several suitable alterations in starch molecules, which promoted the transglycosylation efficiency of the following Gt-GBE treatment. This dual GBE-catalyzed modification process offered an efficient strategy for regulating starch digestibility.

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Influence of microwave treatment on the structure and functionality of pure amylose and amylopectin systems.

Zhong, Y., Tian, Y., Liu, X., Ding, L., Kirkensgaard, J. J. K., Hebelstrup, K., Putaux, J. L. & Blennow, A. (2021). Food Hydrocolloids, 119, 106856.

Pure granular amylose (AM) and pure granular amylopectin (waxy) starch (AP) granules have the high nutritional value in food industry. Effects of microwave treatment (400 W/g DW, 1-8 min) on the structure and properties of transgenic AM granules and AP granules were investigated in direct comparison. Microwave treatment, especially during the first 3 min, decreased the molecular weight of molecules in both the AM and the AP samples. The crystallinity of AM starch initially increased from 15.6% to 20.6%, which was associated with the formation of new Vh-type crystals. After that, crystallinity decreased alongside to 11.3% with the complete disruption of B-type crystals. In contrast, the crystallinity of AP starch initially decreased from 18.9% to 10.8% followed by an increase to 20.0%. Upon prolonged treatment of AM granules, the resistant starch and water solubility was significantly increased. Our data demonstrate notable different microwave-dependent reorganization patterns for pure granular AM and AP molecules as native granular systems, which is helpful to the improvement of functionality of these two starches.

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Structural elements determining the transglycosylating activity of glycoside hydrolase family 57 glycogen branching enzymes.

Xiang, G., Leemhuis, H. & van der Maarel, M. (2021). Authorea Preprints, In Press.

Glycoside hydrolase family 57 glycogen branching enzymes (GH57GBE) catalyze the formation of an α-1,6 glycosidic bond between α-1,4 linked glucooliogosaccharides. As an atypical family, a limited number of GH57GBEs have been biochemically characterized so far. This study aimed at acquiring a better understanding of the GH57GBE family by a systematic sequence-based bioinformatics analysis of almost 2500 gene sequences and determining the branching activity of several native and mutant GH57GBEs. A correlation was found in a very low or even no branching activity with the absence of a flexible loop, a tyrosine at the loop tip, and two β-strands.

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New insights into the alleviating role of starch derivatives on dough quality deterioration caused by freeze.

Li, Y., Li, C., Ban, X., Cheng, L., Hong, Y., Gu, Z. & Li, Z. (2021). Food Chemistry, 130240.

The alleviating role of starch derivatives on the quality deterioration of frozen steamed bread dough was investigated in terms of derivative structure, the bread characteristics and dough properties including freezable water contents, yeast activity as well as dough viscoelasticity. The addition of starch derivatives including short-clustered maltodextrin (SCMD), DE2 maltodextrin (MD) and pregelatinized starch (PGS) significantly increased the specific volume and decreased the hardness of steamed bread compared with Control bread after 8-week frozen storage. Lower freezable water content was found in PGS dough than SCMD dough, which was consistent with the results of water absorption index of starch derivatives. The analysis of dough gassing rate and yeast survival ratio demonstrated SCMD could provide more cryoprotection for yeast cells. Meanwhile, a higher elastic module and a more continuous gluten-network structure of SCMD dough were found after 8-week frozen storage. These results indicated starch derivatives especially SCMD were promising to be used as the alternative improvers in frozen dough production.

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A simplified method of determining the internal structure of amylopectin from barley starch without amylopectin isolation.

Zhao, X., Andersson, M. & Andersson, R. (2020). Carbohydrate Polymers, 117503.

To determine the internal structure of barley starch without amylopectin isolation, whole starch was hydrolyzed using β-amylase to remove the linear amylose and obtain β-limit dextrins (β-LDs). The β-LDs were treated with extensive α-amylase to prepare α-limit dextrins (α-LDs), and the α-LDs were further hydrolyzed with β-amylase into building blocks. The chain-length distribution of β-LD and building block composition were analyzed by size-exclusion chromatography and anion-exchange chromatography. The internal structure of the barley whole starches had similar pattern to barley amylopectins analyzed by conventional methods. The starch of barley amo1-mutated varieties contained more short internal B-chains and less long internal B-chains than that of other varieties. The starch from amo1-mutated varieties had more large building blocks than that from waxy varieties. The simplified method presented in this study can effectively characterize starch internal structure that relates to physicochemical properties of starch, although some details of amylopectin structure are not assessable.

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Digestion kinetics of low, intermediate and highly branched maltodextrins produced from gelatinized starches with various microbial glycogen branching enzymes.

Zhang, X., Leemhuis, H. & van der Maarel, M. J. (2020). Carbohydrate Polymers, 247, 116729.

Twenty-four branched maltodextrins were synthesized from eight starches using three thermostable microbial glycogen branching enzymes. The maltodextrins have a degree of branching (DB) ranging from 5 % to 13 %. This range of products allows us to explore the effect of DB on the digestibility, which was quantified under conditions that mimic the digestion process in the small intestine. The rate and extent of digestibility were analyzed using the logarithm of the slope method, revealing that the branched maltodextrins consist of a rapidly and slowly digestible fraction. The amount of slowly digestible maltodextrin increases with an increasing DB. Surprisingly, above 10 % branching the fraction of slowly digestible maltodextrin remains constant. Nevertheless, the rate of digestion of the slowly digestible fraction was found to decline with increasing DB and shorter average internal chain length. These observations increase the understanding of the structural factors important for the digestion rate of branched maltodextrins.

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On the role of the internal chain length distribution of amylopectins during retrogradation: double helix lateral aggregation and slow digestibility.

Roman, L., Yee, J., Hayes, A. M., Hamaker, B., Bertoft, E. & Martinez, M. M. (2020). Carbohydrate Polymers, 246, 116633.

A structure-digestion model is proposed to explain the formation of α-amylase-slowly digestible structures during amylopectin retrogradation. Maize and potato (normal and waxy) and banana starch (normal and purified amylopectin through alcohol precipitation), were analyzed for amylose ratio and size (HPSEC) and amylopectin unit- and internal-chain length distribution (HPAEC). Banana amylopectin (BA), like waxy potato (WP), exhibited a larger number of B3-chains, fewer BS- and Bfp-chains and lower S:L and BS:BL ratios than maize, categorizing BA structurally as type-4. WP exhibited a significantly greater tendency to form double helices (DSC and 13C-NMR) than BA, which was attributed to its higher internal chain length (ICL) and fewer DP6−12-chains. However, retrograded BA was remarkably more resistant to digestion than WP. Lower number of phosphorylated B-chains, more S- and Bfp-chains and shorter ICL, were suggested to result in α-amylase-slowly digestible structures through further lateral packing of double helices (suggested by thermo-rheology) in type-4 amylopectins.

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Cassava Metabolomics and Starch Quality.

Rosado‐Souza, L., David, L. C., Drapal, M., Fraser, P. D., Hofmann, J., Klemens, P. A., et al.  (2019). Current protocols in Plant Biology, 4(4), e20102.

Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient‐depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non‐targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze‐dried material in order to allow processing of field harvest samples that typically will require long‐distance transport.

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Structural characterization of mixed-linkage α-glucans produced by mutants of Lactobacillus reuteri TMW 1.106 dextransucrase.

Münkel, F., Fischer, A., & Wefers, D. (2020). Carbohydrate Polymers, 231, 115697.

Dextrans and other bacterial α-glucans are versatile and structurally diverse polysaccharides which can be enzymatically synthesized by using glucansucrases. By substituting certain amino acids in the active site of these enzymes, the structure of the synthesized polysaccharides can be modified. In this study, such amino acid substitutions were applied (single and combined) to the dextransucrase from Lactobacillus reuteri TMW 1.106 and the structures of the synthesized polysaccharides were subsequently characterized in detail. Besides methylation analysis, α-glucans were hydrolyzed by several glycoside hydrolases and the liberated oligosaccharides were identified by comparison to standard compounds or by isolation and NMR spectroscopic characterization. Furthermore, two-dimensional NMR spectroscopy was used to analyze the untreated polysaccharides. The results demonstrated that structurally different α-glucans were formed, for example different highly O4-branched dextrans or several reuteran-like polymers with varying fine structures. Consequently, mutant Lactobacillus reuteri TMW 1.106 dextransucrases can be used to form structurally unique polysaccharides.

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A novel starch: Characterizations of starches separated from tea (Camellia sinensis (L.) O. Ktze) seed.

Huang, J., Kong, X., Wu, D., Zheng, Z. & Shu, X. (2019). International Journal of Biological Macromolecules, 139, 1085-1091.

The physicochemical, thermal and crystal properties of starches isolated from 3 different tea (Camellia sinensis (L.) O. Ktze) seeds were analyzed in this study. The shape of tea starch granules were flat spherical or oval shape, showed unimodal or bimodal distribution with average size of around 9 μm. Tea starch was typical A-type starch. Apparent amylose contents of three tea seed starches ranged from 27.06% to 33.17%. The chains having degree of polymerization (DP) 13-24 were over 50% of the total detectable chains for tea amylopectin. Peak gelatinization temperature of tea starch ranged from 65 to 77°C and the water solubility reached up to 9.70%. The peak viscosity of tea starches were as high as 5300 cP and final viscosity ranged from 4000 to 6700 cP. The results indicated that tea seed starch had potential as gel reagents and provide some guides for comprehensive utilization of tea starch in food and non-food applications.

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Physiochemical properties of rice with contrasting resistant starch content.

Ding, Y., Huang, J., Zhang, N., Rasmussen, S. K., Wu, D. & Shu, X. (2019). Journal of Cereal Science, 89, 102815.

RS2 and RS3 are two of the main resistant starch types in food, and the primary types found in raw and cooked rice respectively. To elucidate the physiochemical determinants for RS2 and RS3 content in rice, this study investigated the fine amylopectin structure, starch granules morphologies and starch pasting properties of nine different rice accessions. The results revealed that not all the accessions showed a decrease in resistant starch after cooking and cooling. Granular size, paste properties, fb1 and fb3 showed a significant negative correlation with RS3, whereas fa showed a significant positive correlation with RS3. RS2 had no significant correlation with granular size, but did have a significant positive correlation with HPV, CPV and fb2. Different rice types could be distinguished by the fine amylopectin structure of DP6-12 and DP13-24 chains. Taken together fa, fb2 and HPV and CPV values may have the potential to be used as indices for distinguishing or characterising rice with different RS2 and RS3 contents.

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Characterization of the GH13 and GH57 glycogen branching enzymes from Petrotoga mobilis SJ95 and potential role in glycogen biosynthesis.

Zhang, X., Leemhuis, H. & van der Maarel, M. J. (2019). PloS One, 14(7), e0219844.

Glycogen is a highly branched α-glucan polymer widely used as energy and carbon reserve by many microorganisms. The branches are introduced by glycogen branching enzymes (EC, that are classified into glycoside hydrolase families 13 (GH13) and 57 (GH57). Most microorganisms have typically only a single glycogen branching enzyme (gbe) gene. Only a few microorganisms carry both GH13 and GH57 gbe genes, such as Petrotoga mobilis and Mycobacterium tuberculosis. Here we report the basic characteristics of the GH13 and GH57 GBE of Pmobilis, both heterologously expressed in Ecoli. The GH13 GBE has a considerably higher branching activity towards the linear α-glucan amylose, and produces a highly branched α-glucan with a high molecular weight which is very similar to glycogen. The GH57 GBE, on the contrary, makes a much smaller branched α-glucan. While the GH13 GBE acts as a classical glycogen branching enzyme involved in glycogen synthesis, the role of GH57 GBE remains unclear.

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Comparison of molecular structure of oca (Oxalis tuberosa), potato, and maize starches.

Zhu, F. & Cui, R. (2019). Food Chemistry, 296, 116-122.

Oca (Oxalis tuberosa) is an underutilized species and represents a novel starch source. Composition and structure of starches from tubers of two commercial oca varieties grown in New Zealand were compared to those of normal maize and potato starches. The phosphorus content of oca starch was ∼60% of that of potato starch. The amylose content of oca starch (∼21%) was lower than that of maize and potato starches (concanavalin A precipitation method). The fine structure of oca amylopectin was much more similar to that of potato amylopectin than to that of maize amylopectin. Oca amylopectin had a shorter internal chain length and less fingerprint B-chains than potato amylopectin. The two oca starches were structurally and compositionally similar. Oca starch granules had a volume moment mean size of 34.5 μm and B-type polymorph. Comparative analysis suggested that oca starch has the potential to be developed as a novel starch source.

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Synthesis of highly branched α-glucans with different structures using GH13 and GH57 glycogen branching enzymes.

Zhang, X., Leemhuis, H. & van der Maarel, M. J. (2019). Carbohydrate Polymers, 216, 231-237.

Glycogen branching enzymes (GBEs) convert starch into branched α-glucan polymers. To explore if the amylose content of substrates effects the structure of the branched α-glucans, mixtures of amylose and amylopectin were converted by four thermophilic GBEs. The degree of branching and molecular weight of the products increased with an increasing percentage of amylose with the GH57 GBEs of Thermus thermophilus and Thermococcus kodakarensis, and the GH13 GBEs of Rhodothermus marinus and Petrotoga mobilis. The only exception is that the degree of branching of the Petrotoga mobilis GBE products is not influenced by the amylose content. A second difference is the relatively high hydrolytic activity of two GH57 GBEs, while the two GH13 GBEs have almost no hydrolytic activity. Moreover, the two GH13 GBEs synthesize branched α-glucans with a narrow molecular weight distribution, while the two GH57 GBEs products consist of two or three molecular weight fractions.

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Organelle DNA contents and starch accumulation in potato tubers.

Niu, S., Zhang, G., Li, X., Haroon, M., Si, H., Fan, G. & Li, X. Q. (2019). Theoretical and Applied Genetics, 132(1), 205-216.

Starch is the main dry matter component of various staple food crops, including potato. Starch synthesis and accumulation is in plastids, uses sugar, consumes cellular energy, and requires active expression of starch synthesis genes. We hypothesized that the plastid/nuclear DNA ratios and mitochondrial/nuclear DNA ratios are involved in this accumulation. We analyzed the dry mater, starch, plastid DNA, mitochondrial DNA, and nuclear DNA in tuber stem ends and tuber bud ends in two potato cultivars and verified the results using whole tubers in nine potato cultivars. Dry matter contents (DMC) and organelle/nuclear DNA ratios increased rapidly during tuber bulking. DMC and starch contents were greater at the tuber stem ends than at the tuber bud ends. Both the comparisons between tuber ends and among whole tubers indicated that DMC and starch contents were positively correlated with both plastid/nuclear DNA ratios and mitochondrial/nuclear DNA ratios. The results suggest that pt/nuc and mt/nuc DNA ratios are important and may serve as a biomarker in selection, genetic engineering, and cytoplasm manipulation, for dry matter and starch accumulation in potato.

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Molecular structure of Maori potato starch.

Zhu, F. & Hao, C. (2018). Food Hydrocolloids, 80, 206-211.

New Zealand Maori potatoes (Taewa) represent unique genetic resources for potato quality, though they are much underutilized. In this report, the composition and molecular structure of starches from 5 Maori potato varieties were studied. In particular, the internal unit chain composition of the amylopectins in the form of β-limit dextrins were highlighted. Starches from a commercial modern potato variety and a maize variety with normal amylose contents were employed for comparison. Genetic diversity in the amylose (e.g., 22.6% in Moemoe to 28.6% in Turaekuri) and phosphorus (5.4 mg/100 g in Turaekuri to 7.0 mg/100 g in Kowiniwini) contents as well as the molecule structure of the starches (e.g., external chain length of amylopectin ranged from 13.0 glucosyl residues in Turaekuri to 15.8 glucosyl residues in Karuparera) has been revealed. Maori potato amylopectins have the highest amount of long unit and internal chains and the lowest amount of these chains among amylopectins from different sources. Overall, Maori potato starch appeared to be structurally and compositionally similar to modern potato starch.

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Effect of hydroxypropylation and beta‐amylase treatment on complexation of debranched starch with naringenin.

Gonzalez, A., Wang, Y. J., Staroszczyk, H., Brownmiller, C. & Lee, S. O. (2018). Starch‐Stärke, In Press.

Naringenin exhibits many health benefits but it has limited water solubility and consequently low bioavailability. The objective of this study was to investigate the effect of hydroxypropylation and enzymatic treatments on starch complexation with naringenin. Potato starch and Hylon VII were hydroxypropylated to two substitution degrees and then debranched or debranched/β-amylase treated prior to complexing with naringenin. Both soluble and insoluble complexes were recovered and characterized. An increase in hydroxypropylation level improved recovery of soluble complexes, while total recovery remained unchanged; the β-amylase treatment further increased soluble complex recovery. For the same treatment, the naringenin content was greater in Hylon VII complexes (6.72-15.15mg/g) than in potato starch complexes (2.45-11.18 mg/g). Insoluble complexes comprised greater naringenin contents (3.91-15.15 mg/g) compared to soluble counterparts (2.45-9.43 mg/g). All complexes exhibited a mixture of B+V X-ray diffraction pattern. This work is the first one to demonstrate that hydroxypropylated starch formed complexes with naringenin, and an appropriate level of beta-amylase hydrolysis further improved their complexation.

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Safety Information
Symbol : GHS08
Signal Word : Danger
Hazard Statements : H334
Precautionary Statements : P261, P284, P304+P340, P342+P311, P501
Safety Data Sheet
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