Characterization of the Covalent Complex Derived from Interaction between Milk Thistle Protein and Rosemary Phenolic Extract

Document Type : Complete scientific research article

Authors

1 Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran

2 M.Sc. student, Deptment of Food Science and Technology, Gorgan University of Agricultural Science and Natural Resources

3 Professor, Department of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources

4 Ph.D. student, Dept. of Food Science and Technology, Gorgan University of Agricultural Science and Natural Resources

Abstract

Background and Objective: Plant protein concentrates, owing to their high protein content and low fat levels, are considered suitable matrices for targeted chemical modifications and for investigating molecular interactions with bioactive compounds. Proteins derived from milk thistle seeds (Silybum marianum L.), with a balanced amino acid profile and favorable structural characteristics, exhibit a high potential for interaction with phenolic compounds from medicinal plants—interactions that can lead to enhanced antioxidant activity and improved stability of inherently unstable phenolic compounds. Rosemary (Rosmarinus officinalis) is a rich source of phenolic compounds with strong antioxidant activity and has the ability to form stable covalent complexes with proteins. The main objective of this study was to investigate the formation of covalent complexes between milk thistle protein concentrate and rosemary extract and to evaluate the effects of this interaction on antioxidant activity and phenolic contents of complex.
Materials and Methods: Milk thistle seeds and rosemary plants were collected and dried. The seeds were defatted, and a protein concentrate was prepared. The phenolic extract of rosemary was obtained using a hydroalcoholic solvent extraction and subsequently mixed with the protein concentrate at different concentrations (1–6%) to induce the formation of covalent complexes. Total phenolic content, flavonoid content, total antioxidant capacity, and ferric reducing power of the samples were determined using the Folin–Ciocalteu colorimetric method, spectrophotometric assays, and the phosphomolybdenum method, respectively. Statistical analysis was performed using one-way ANOVA followed by Duncan’s multiple range test at a 95% confidence level.
Results: Chemical composition analysis revealed that the milk thistle protein concentrate contained 53.47% protein and 2.04% fat. Rosemary extract exhibited considerable amounts of phenolic compounds (56.50 mg GAE/g) and flavonoids (9 mg QE/g). The formation of covalent complexes resulted in a significant increase in phenolic content; as the rosemary extract concentration increased from 1% to 6%, total phenolics rose from 233.31 mg GAE/g to 900.153 mg GAE/g. Moreover, the total antioxidant capacity of the system increased in a concentration-dependent manner, with the lowest activity observed in the pure protein concentrate and the highest activity in the complex containing 6% rosemary extract. These findings indicate the successful formation of stable phenolic–protein complexes and their positive effect on antioxidant activity.
Conclusion: The formation of covalent complexes between milk thistle protein concentrate and rosemary phenolic extract is considered an effective approach for increasing total phenolic content and enhancing the antioxidant capacity of the system. The results showed that increasing the concentration of rosemary extract led to a significant increase in total phenolic compounds and antioxidant activity, which indicates the occurrence of effective and substantial interactions between the protein matrix and phenolic compounds. Based on the present findings, this system possesses appropriate potential for use as a plant-based functional additive in food formulations. The results of this study are consistent with previously reported studies on protein–phenolic interactions.

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Food Processing and Preservation Journal
Volume 18, Issue 2
June 2026
Pages 67-82

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