Preparation and emulsifying properties of flaxseed gum modified alfalfa leaf protein
Medicago sativa L. is a high-quality legume forage that is favored by domestic and foreign researchers due to its rich nutritional content, excellent quality, and high protein content. Alfalfa has high yield and low market price, making it a high-quality natural resource. In addition, the crude leaf protein content in alfalfa leaves can reach about 70%, and there are abundant types of amino acids, including 40.6% essential amino acids for the human body. Due to its superior functional characteristics compared to animal protein, it is often used as an auxiliary feed for animals. However, the functional properties of alfalfa leaf protein concentrates (ALPCs) are limited, and their emulsifying activity is not significant. When applied, it is often necessary to add sugar substances with high emulsifying activity to improve their utilization value in food, medicine, and other fields. Glycosylation modification of ALPCs to improve their emulsifying properties has potential utilization value. At present, commonly used protein emulsification modification methods include chemical modification, physical modification, and enzyme modification. The glycosylation modification method in chemical modification is a mild and green protein modification method. By controlling time, temperature, pH, and the degree of grafting reaction between proteins and sugars during heating, the aim is to achieve product modification. Previous studies have shown that glycosylation modification can improve protein emulsifying properties, emulsifying stability, and antioxidant capacity. At present, research on ALPCs only focuses on product preparation and antioxidant activity, and there have been no reports on their emulsifying properties and emulsifying stability. Therefore, in order to compensate for the lack of emulsifying properties of ALPCs, this study optimized the preparation of the composite using glycosylation modification method and studied its emulsifying properties.

Research has reported that polysaccharides can improve the emulsifying properties of proteins. Flaxseed gum (FG), also known as Frank gum, is a natural new hydrophilic colloid composed of acidic and neutral polysaccharides. Research has shown that hydrophilic colloids contain a certain amount of binding proteins, which enable FG to possess good emulsifying, thickening, and foaming properties. In addition, FG is a green, healthy, and safe food specific additive recognized by the National Green Food Development Center. The combination of FG and ALPCs glycosylation has shown great potential in improving protein properties and has become one of the research hotspots in the food industry.

The commonly used glycosylation modification methods currently include dry modification and wet modification. The dry reaction temperature is limited, generally between 60-65 ℃, while the wet reaction temperature is more extensive, generally including low-temperature reactions (60-62 ℃) and high-temperature reactions (100-120 ℃). Due to the advantages of short reaction period and sufficient contact with substrates, the wet heat reaction was modified under wet heat and high temperature conditions in this experiment. The composite was prepared using response surface optimization, and the glycosylation modification of ALPCs was studied using FG. The emulsifying properties of ALPCs FG were measured, and the functional group changes and microstructure of ALPCs FG were characterized, providing theoretical reference for the development of ALPCs resources and the study of emulsifying properties of ALPCs FG.

This study optimized the preparation of ALPCs FG covalent complexes using the BOX Behnken response surface methodology. The optimal conditions were pH 8.0, temperature 90 ℃, reaction time 124 min, and substrate ratio [m (alfalfa leaf protein): m (flaxseed gum)] 1:1. The best emulsification was achieved under these conditions. Under this condition, the emulsifying property of the product can reach 200.78 ㎡/g, which is 12.20% higher than that of unmodified protein. The emulsion stability increased by 31.83%, and the grafting degree under optimal conditions reached 23.9%. Characterization analysis of its ALPCs FG structure revealed the presence of sugar ring structures in the infrared spectrum, indicating successful grafting of FG onto alfalfa leaf protein; The particle size analysis results indicate that glycosylation reaction leads to an increase in particle size; The X-ray diffraction pattern indicates a change in the grain size of the composite, indicating the occurrence of glycosylation reaction; The scanning electron microscopy results indicate that the surface density of the composite increases and the structure is amorphous. Based on the above analysis, glycosylation reaction can significantly improve the emulsifying properties and emulsifying stability of ALPCs FG