Foodborne pathogen Bacillus cereus is widely distributed in dairy products and the processing environment. Beyond its threat to human health, the production of thermostable protease also poses a significant challenge to the dairy industry, particularly in pasteurised or ultra-high-temperature (UHT) processed milk products. Despite this, our understanding of dairy spoilage caused by B. cereus is very limited. This study aimed to evaluate the spoilage potential of a thermostable protease produced by B. cereus 12–1 in fluid milk. Specifically, crude protease characterisation, including optimum pH and temperature, the influence of Ca 2+ on enzyme activity and heat stability of protease, was studied. Moreover, the effects of this protease on the quality of both whole and skim UHT milk were evaluated, including zeta potential and particle size of milk, hydrolysis of milk protein, release of free amino acids and change of milk microstructure. This crude protease has relatively good stability at wide-ranging pH values from 5.0 to 10.0 (79.67–166.67 U/mL) and temperatures from 20°C to 70°C (92.67–165 U/mL), and maintained stability of 46.04% and 38.31% after heat treatment by 65°C/30 min or 100°C/10 min. Spoilage patterns in skim and whole UHT milk differ significantly due to variations in fat content. The protease caused protein hydrolysis in whole milk (15.25%) and skim milk (17.09%), effectively hydrolysed κ-casein and β-casein, caused the release of free amino acids (Lys, Val, Tyr, Phe, Ile, Met and Leu) in UHT milk and significantly increased particle size (increase by 940 nm for skim milk and 1319 nm for whole milk), and reduced zeta potential (reduction by 4 mV for skim milk and 3.83 mV for whole milk) of UHT milk. Results indicate the spoilage potential of crude protease from B. cereus and highlight the need to prevent dairy spoilage caused by B. cereus .
Shan Li,Haifeng Wu,Yuanxi Liu,Baoli Zhang,Shichao Xu,Zhen-Gang Wang
Enzymes rely on protein folding to create functional sites essential for their catalytic activity. Reproducing enzymatic active sites in artificial systems has been difficult due to the intricate folding patterns that enzymes demonstrate. Inspired by natural systems, we developed a supramolecular copper-cluster-dependent catechol oxidase. This system is constructed through the self-assembly of flavin adenine dinucleotide (FAD) with fluorenylmethyloxycarbonyl-modified amino acids (e.g., Fmoc-lysine) and Cu2+ ions. Fmoc-amino acids self-associate through fluorenyl stacking, facilitating the formation of oxidase-mimetic copper clusters. FAD self-assembles with Fmoc-amino acids via aromatic stacking, enabling the incorporation of adenine and amino acid groups to form a coordination sphere around Cu2+. The self-assembly of FAD with Fmoc-amino acids enhances the oxidase-like activity of Cu2+ by over 100-fold. It is estimated that each copper can catalyze the transformation of ca. 80 substrate molecules within 15 min. The catalyst demonstrated thermophilic properties and excellent storage stability. When exposed to 460 nm light, the activity of the FAD/Fmoc-K/Cu2+ complex is further enhanced, potentially due to cooperative interaction between FAD and lumichrome generated from FAD photodegradation. These findings have implications for the design of advanced enzyme-mimetic catalysts.
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