Optimising Enzymatic Cross-Linking: Impact on Physicochemical and Functional Properties of Lupin Flour and Soy Protein Isolate
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Proximate Analysis of Lupin Flour and Soy Protein Isolate
2.3. Enzyme Treatment of Lupin and Soy Protein
2.3.1. Sample and Reagent Preparation
2.3.2. Optimisation of Protein Cross-Linking Reactions Between LF and SPI
- Experiment 1 (incubation time): Reaction time of 1, 5, 10, and 20 h at pH 7 and 30 °C.
- Experiment 2 (pH variation): Reactions at pH levels 4, 5, 6, 7, and 8 for 1 h at 30 °C.
- Experiment 3 (ferulic acid addition, enzyme concentration, and incubation temperature): Enzyme concentration ranged between 71.25, 142.5, and 285 U/100 mg protein for LR and 1.25, 2.5, 5, and 10 U/100 mg protein for TG. Samples were incubated for 1 h and 20 h at 20 °C and 30 °C at pH 6. Notably, ferulic acid (25 mM) was added as a catalyst in LR-treated samples.
2.3.3. SDS-PAGE
2.4. Physicochemical Properties
2.4.1. ζ-Potential
2.4.2. Particle Size Distribution
2.4.3. Morphology
2.5. Functional Properties
2.5.1. Protein Solubility
2.5.2. Emulsifying Properties
2.5.3. Foaming Properties
2.6. Statistical Analysis
3. Results and Discussion
3.1. Proximate Analysis
3.2. Effects of Reaction Conditions on Lupin and Soy Protein Cross-Linking
3.2.1. Protein Profile of Lupin and Soy Proteins
3.2.2. Effects of Varying Reaction Conditions on Lupin and Soy Protein Cross-Linking
Incubation Time
pH
Ferulic Acid Addition, Enzyme Concentration and Incubation Temperature
Ferulic Acid and LR-Induced Cross-Linking
Enzyme Concentration and Incubation Temperature
3.3. Physicochemical Properties
3.3.1. ζ-Potential
3.3.2. Particle Size Distribution
3.3.3. Morphology
3.4. Functional Properties
3.4.1. Protein Solubility
3.4.2. Emulsifying Properties
Emulsifying Ability
Emulsion Stability
3.4.3. Foaming Properties
Foaming Ability
Foaming Stability
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Day, L. Proteins from land plants—Potential resources for human nutrition and food security. Trends Food Sci. Technol. 2013, 32, 25–42. [Google Scholar] [CrossRef]
- Nikbakht Nasrabadi, M.; Sedaghat Doost, A.; Mezzenga, R. Modification approaches of plant-based proteins to improve their techno-functionality and use in food products. Food Hydrocoll. 2021, 118, 106789. [Google Scholar] [CrossRef]
- Ma, W.; Wang, T.; Wang, J.; Wu, D.; Wu, C.; Du, M. Enhancing the thermal stability of soy proteins by preheat treatment at lower protein concentration. Food Chem. 2020, 306, 125593. [Google Scholar] [CrossRef] [PubMed]
- Malik, M.A.; Sharma, H.K.; Saini, C.S. Effect of gamma irradiation on structural, molecular, thermal and rheological properties of sunflower protein isolate. Food Hydrocoll. 2017, 72, 312–322. [Google Scholar] [CrossRef]
- Heck, T.; Faccio, G.; Richter, M.; Thöny-Meyer, L. Enzyme-catalyzed protein crosslinking. Appl. Microbiol. Biotechnol. 2013, 97, 461–475. [Google Scholar] [CrossRef]
- Gerrard, J.A. Protein–protein crosslinking in food: Methods, consequences, applications. Trends Food Sci. Technol. 2002, 13, 391–399. [Google Scholar] [CrossRef]
- Santoso, T.; Ho, T.M.; Vinothsankar, G.; Jouppila, K.; Chen, T.; Owens, A.; Lazarjani, M.P.; Farouk, M.M.; Colgrave, M.L.; Otter, D.; et al. Effects of Laccase and Transglutaminase on the Physicochemical and Functional Properties of Hybrid Lupin and Whey Protein Powder. Foods 2024, 13, 2090. [Google Scholar] [CrossRef] [PubMed]
- Konieczny, D.; Stone, A.K.; Korber, D.R.; Nickerson, M.T.; Tanaka, T. Physicochemical properties of enzymatically modified pea protein-enriched flour treated by different enzymes to varying levels of hydrolysis. Cereal Chem. 2020, 97, 326–338. [Google Scholar] [CrossRef]
- Chen, C.; Zhang, C.; Zhang, R.; Ju, X.; He, R.; Wang, Z. Enzyme-catalyzed acylation improves gel properties of rapeseed protein isolate. J. Sci. Food Agric. 2020, 100, 4182–4189. [Google Scholar] [CrossRef]
- Isaschar-Ovdat, S.; Fishman, A. Crosslinking of food proteins mediated by oxidative enzymes–a review. Trends Food Sci. Technol. 2018, 72, 134–143. [Google Scholar] [CrossRef]
- Buchert, J.; Ercili Cura, D.; Ma, H.; Gasparetti, C.; Monogioudi, E.; Faccio, G.; Mattinen, M.; Boer, H.; Partanen, R.; Selinheimo, E.; et al. Crosslinking Food Proteins for Improved Functionality. Annu. Rev. Food Sci. Technol. 2010, 1, 113–138. [Google Scholar] [CrossRef] [PubMed]
- Sato, A.C.K.; Perrechil, F.A.; Costa, A.A.S.; Santana, R.C.; Cunha, R.L. Cross-linking proteins by laccase: Effects on the droplet size and rheology of emulsions stabilized by sodium caseinate. Food Res. Int. 2015, 75, 244–251. [Google Scholar] [CrossRef] [PubMed]
- Gouseti, O.; Larsen, M.E.; Amin, A.; Bakalis, S.; Petersen, I.L.; Lametsch, R.; Jensen, P.E. Applications of Enzyme Technology to Enhance Transition to Plant Proteins: A Review. Foods 2023, 12, 2518. [Google Scholar] [CrossRef] [PubMed]
- Schlangen, M.; Ribberink, M.A.; Taghian Dinani, S.; Sagis, L.M.C.; van der Goot, A.J. Mechanical and rheological effects of transglutaminase treatment on dense plant protein blends. Food Hydrocoll. 2023, 136, 108261. [Google Scholar] [CrossRef]
- Herz, E.; Herz, L.; Dreher, J.; Gibis, M.; Ray, J.; Pibarot, P.; Schmitt, C.; Weiss, J. Influencing factors on the ability to assemble a complex meat analogue using a soy-protein-binder. Innov. Food Sci. Emerg. Technol. 2021, 73, 102806. [Google Scholar] [CrossRef]
- Mattinen, M.-L.; Hellman, M.; Permi, P.; Autio, K.; Kalkkinen, N.; Buchert, J. Effect of Protein Structure on Laccase-Catalyzed Protein Oligomerization. J. Agric. Food Chem. 2006, 54, 8883–8890. [Google Scholar] [CrossRef]
- Kishimoto, T.; Hiyama, A.; Toda, H.; Urabe, D. Effect of pH on the Dehydrogenative Polymerization of Monolignols by Laccases from Trametes versicolor and Rhus vernicifera. ACS Omega 2022, 7, 9846–9852. [Google Scholar] [CrossRef]
- Yang, W.Y.; Min, D.Y.; Wen, S.X.; Jin, L.; Rong, L.; Tetsuo, M.; Bo, C. Immobilization and characterization of laccase from Chinese Rhus vernicifera on modified chitosan. Process Biochem. 2006, 41, 1378–1382. [Google Scholar] [CrossRef]
- Tlaiaa, Y.S.; Sahar, I.; M-Ridha, M.J. Evaluation the Properties of Purified Laccase Extracted From Some Local Plants Under the Optimum Conditions. Iraqi J. Agric. Sci. 2023, 54, 1101–1112. [Google Scholar] [CrossRef]
- Wang, H.; You, S.; Wang, W.; Zeng, Y.; Su, R.; Qi, W.; Wang, K.; He, Z. Laccase-catalyzed soy protein and gallic acid complexation: Effects on conformational structures and antioxidant activity. Food Chem. 2022, 375, 131865. [Google Scholar] [CrossRef]
- Kieserling, H.; de Bruijn, W.J.C.; Keppler, J.; Yang, J.; Sagu, S.T.; Güterbock, D.; Rawel, H.; Schwarz, K.; Vincken, J.-P.; Schieber, A.; et al. Protein–phenolic interactions and reactions: Discrepancies, challenges, and opportunities. Compr. Rev. Food Sci. Food Saf. 2024, 23, e70015. [Google Scholar] [CrossRef] [PubMed]
- AOAC International. Official Methods of Analysis of AOAC International; AOAC International: Gaithersburg, MD, USA, 2019. [Google Scholar]
- Chen, C.-C.; Chen, L.-Y.; Chan, D.-S.; Chen, B.-Y.; Tseng, H.-W.; Hsieh, J.-F. Influence of Microbial Transglutaminase on Physicochemical and Cross-Linking Characteristics of Individual Caseins. Molecules 2020, 25, 3992. [Google Scholar] [CrossRef]
- Le, T.T.; Nielsen, S.D.; Villumsen, N.S.; Kristiansen, G.H.; Nielsen, L.R.; Nielsen, S.B.; Hammershøj, M.; Larsen, L.B. Using proteomics to characterise storage-induced aggregates in acidic whey protein isolate drinks. Int. Dairy J. 2016, 60, 39–46. [Google Scholar] [CrossRef]
- Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [Google Scholar] [CrossRef] [PubMed]
- Ho, T.M.; Zhu, J.; Bansal, N.; Boyce, M.C.; Le, T.T. Effect of pH and heat treatment on physicochemical and functional properties of spray-dried whey protein concentrate powder. Int. Dairy J. 2021, 119, 105063. [Google Scholar] [CrossRef]
- Liang, J.-N.; Nargotra, P.; Li, X.-Y.; Sharma, V.; Hsieh, S.-L.; Tsai, Y.-H.; Liu, Y.-C.; Huang, C.-Y.; Kuo, C.-H. Evaluation of Wheat Noodles Supplemented with Soy Protein Isolate for Nutritional, Textural, Cooking Attributes and Glycemic Index. Appl. Sci. 2023, 13, 7772. [Google Scholar] [CrossRef]
- Monteiro, S.R.; Lopes-da-Silva, J.A. Critical evaluation of the functionality of soy protein isolates obtained from different raw materials. Eur. Food Res. Technol. 2019, 245, 199–212. [Google Scholar] [CrossRef]
- Hall, R.S.; Thomas, S.J.; Johnson, S.K. Australian sweet lupin flour addition reduces the glycaemic index of a white bread breakfast without affecting palatability in healthy human volunteers. Asia Pac. J. Clin. Nutr. 2005, 14, 91. [Google Scholar] [PubMed]
- Jayasena, V.; Chih, H.J.; Nasar-Abbas, S. Efficient isolation of lupin protein. Food Aust. 2011, 63, 306–309. [Google Scholar]
- Wang, T.; Johnson, L.; Myers, D. Value-added products from extruding-expelling of soybeans. In Soybeans as Functional Foods and Ingredients; Liu, K., Ed.; AOCS Press: Champaign, IL, USA, 2004; pp. 73–100. [Google Scholar] [CrossRef]
- Jiang, J.; Chen, J.; Xiong, Y.L. Structural and Emulsifying Properties of Soy Protein Isolate Subjected to Acid and Alkaline pH-Shifting Processes. J. Agric. Food Chem. 2009, 57, 7576–7583. [Google Scholar] [CrossRef]
- Olukomaiya, O.O.; Adiamo, O.Q.; Fernando, W.C.; Mereddy, R.; Li, X.; Sultanbawa, Y. Effect of solid-state fermentation on proximate composition, anti-nutritional factor, microbiological and functional properties of lupin flour. Food Chem. 2020, 315, 126238. [Google Scholar] [CrossRef] [PubMed]
- Tang, Q.; Roos, Y.H.; Miao, S. Plant Protein versus Dairy Proteins: A pH-Dependency Investigation on Their Structure and Functional Properties. Foods 2023, 12, 368. [Google Scholar] [CrossRef]
- Chen, N.; Zhao, M.; Chassenieux, C.; Nicolai, T. Data on the characterization of native soy globulin by SDS-Page, light scattering and titration. Data Brief 2016, 9, 749–752. [Google Scholar] [CrossRef]
- Barać, M.; Pešić, M.; Žilić, S.; Srebrić, M.; Drinić, S. Identification of soluble protein fractions and their subunits in soybeans with black and yellow kernel coat. In Proceedings of the 46th Croatian and 6th International Symposium on Agriculture, Opatija, Croatia, 14–18 February 2011; pp. 681–685. [Google Scholar]
- Dumitraşcu, L.; Stănciuc, N.; Grigore-Gurgu, L.; Aprodu, I. Spectroscopic and molecular modeling investigations on heat induced behaviour of soy proteins. Emir. J. Food Agric. 2019, 31, 569–579. [Google Scholar] [CrossRef]
- Liu, S.; Zhou, R.; Tian, S.; Gai, J. A study on subunit groups of soybean protein extracts under SDS-PAGE. J. Am. Oil Chem. Soc. 2007, 84, 793–801. [Google Scholar] [CrossRef]
- Rumiyati, R.; James, A.P.; Jayasena, V. Effect of germination on the nutritional and protein profile of Australian sweet lupin (Lupinus angustifolius L.). Food Nutr. Sci. 2012, 3, 621–626. [Google Scholar] [CrossRef]
- Devkota, L.; Kyriakopoulou, K.; Fernandez, D.; Bergia, R.; Dhital, S. Techno-functional and rheological characterization of protein isolates from Australian lupin species as affected by processing conditions. Int. J. Food Sci. Technol. 2023, 59, 774–784. [Google Scholar] [CrossRef]
- Sakai, K.; Sato, Y.; Okada, M.; Yamaguchi, S. Improved functional properties of meat analogs by laccase catalyzed protein and pectin crosslinks. Sci. Rep. 2021, 11, 16631. [Google Scholar] [CrossRef]
- Gorissen, S.H.M.; Crombag, J.J.R.; Senden, J.M.G.; Waterval, W.A.H.; Bierau, J.; Verdijk, L.B.; van Loon, L.J.C. Protein content and amino acid composition of commercially available plant-based protein isolates. Amino Acids 2018, 50, 1685–1695. [Google Scholar] [CrossRef]
- Paananen, A.; Ercili-Cura, D.; Saloheimo, M.; Lantto, R.; Linder, M.B. Directing enzymatic cross-linking activity to the air–water interface by a fusion protein approach. Soft Matter 2013, 9, 1612–1619. [Google Scholar] [CrossRef]
- Vijayan, P.; Song, Z.; Toy, J.Y.H.; Yu, L.L.; Huang, D. Effect of transglutaminase on gelation and functional proteins of mung bean protein isolate. Food Chem. 2024, 454, 139590. [Google Scholar] [CrossRef] [PubMed]
- Nivala, O.; Mäkinen, O.E.; Kruus, K.; Nordlund, E.; Ercili-Cura, D. Structuring colloidal oat and faba bean protein particles via enzymatic modification. Food Chem. 2017, 231, 87–95. [Google Scholar] [CrossRef]
- Liu, C.; Damodaran, S.; Heinonen, M. Effects of microbial transglutaminase treatment on physicochemical properties and emulsifying functionality of faba bean protein isolate. LWT 2019, 99, 396–403. [Google Scholar] [CrossRef]
- Ma, K.K.; Greis, M.; Lu, J.; Nolden, A.A.; McClements, D.J.; Kinchla, A.J. Functional Performance of Plant Proteins. Foods 2022, 11, 594. [Google Scholar] [CrossRef] [PubMed]
- Liu, Y.; Zhang, Y.; Guo, Z.; Wang, C.; Kang, H.; Li, J.; Wang, W.; Li, Y.; Lu, F.; Liu, Y. Enhancing the functional characteristics of soy protein isolate via cross-linking catalyzed by Bacillus subtilis transglutaminase. J. Sci. Food Agric. 2021, 101, 4154–4160. [Google Scholar] [CrossRef]
- Xiang, H.; Sun-waterhouse, D.; Cui, C.; Wang, W.; Dong, K. Modification of soy protein isolate by glutaminase for nanocomplexation with curcumin. Food Chem. 2018, 268, 504–512. [Google Scholar] [CrossRef]
Experiments | Variation | Time (h) | pH | Temperature (°C) | Catalyst | Enzyme Concentration (U/100 mg Protein) |
---|---|---|---|---|---|---|
Experiment 1 | Incubation time | 1, 5, 10, and 20 | 7 | 30 | - | LR: 142.5 TG: 10 |
Experiment 2 | pH | 1 | 4–8 | 30 | - | LR: 285 TG: 10 |
Experiment 3 | Ferulic acid addition, enzyme concentration, and incubation temperature | 1 and 20 | 6 | 20 and 30 | 25 mM ferulic acid (LR only) | LR: 71.25, 142.5, and 285 TG 1.25, 2.5, 5, and 10 |
Attributes | LF | SPI |
---|---|---|
Moisture (%) | 7.10 ± 0.30 | 6.50 ± 0.10 |
Ash (%) | 2.60 ± 0.04 | 4.90 ± 0.02 |
Fat (%) | 6.00 ± 0.02 | 0.09 ± 0.03 |
Protein (%) | 43.70 ± 0.90 | 87.60 ± 0.30 |
Carbohydrate (%) | 40.60 ± 0.80 | 0.90 ± 0.20 |
pH | 6.19 ± 0.02 | 7.72 ± 0.02 |
Sample Name | D[4,3], µm | D[3,2], µm |
---|---|---|
LF | 46.2 ± 6.2 d | 12.4 ± 0.5 f |
SPI | 27.9 ± 4.4 d | 14.3 ± 0.6 d |
LS-CT-1H-20C | 28.2 ± 0.3 d | 14.1 ± 0.1 de |
LS-LR-1H-20C | 47.8 ± 1.9 b | 11.4 ± 0.1 g |
LS-LR-FeA-1H-20C | 58.3 ± 6.4 a | 17 ± 0.1 b |
LS-CT-20H-20C | 36.4 ± 5.1 c | 15 ± 0.7 c |
LS-LR-20H-20C | 24.7 ± 0.5 d | 9.9 ± 0 h |
LS-LR-FeA-20H-20C | 61.2 ± 7.7 a | 13.7 ± 0.5 de |
LS-CT-1H-30C | 35.9 ± 0.9 c | 13.5 ± 0.1 e |
LS-TG-1H-30C | 42.4 ± 3.7 bc | 17.9 ± 0.3 a |
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Santoso, T.; Al-Shaikhli, Y.; Ho, T.M.; Rajapakse, M.; Le, T.T. Optimising Enzymatic Cross-Linking: Impact on Physicochemical and Functional Properties of Lupin Flour and Soy Protein Isolate. Foods 2025, 14, 1976. https://doi.org/10.3390/foods14111976
Santoso T, Al-Shaikhli Y, Ho TM, Rajapakse M, Le TT. Optimising Enzymatic Cross-Linking: Impact on Physicochemical and Functional Properties of Lupin Flour and Soy Protein Isolate. Foods. 2025; 14(11):1976. https://doi.org/10.3390/foods14111976
Chicago/Turabian StyleSantoso, Teguh, Yusur Al-Shaikhli, Thao M. Ho, Mishenki Rajapakse, and Thao T. Le. 2025. "Optimising Enzymatic Cross-Linking: Impact on Physicochemical and Functional Properties of Lupin Flour and Soy Protein Isolate" Foods 14, no. 11: 1976. https://doi.org/10.3390/foods14111976
APA StyleSantoso, T., Al-Shaikhli, Y., Ho, T. M., Rajapakse, M., & Le, T. T. (2025). Optimising Enzymatic Cross-Linking: Impact on Physicochemical and Functional Properties of Lupin Flour and Soy Protein Isolate. Foods, 14(11), 1976. https://doi.org/10.3390/foods14111976