The pleasant taste of edible mushrooms, which is attributed to their high protein content, makes them an attractive source for the production of protein hydrolysates with good taste properties. In the present work, different mushroom protein hydrolysates were produced from shiitake, oyster, bunashimeji and enoki mushrooms using stem bromelain hydrolysis at 0.5% (m/m) enzyme/substrate ratio at pH=6.5 and 40 °C for 20 h. The produced liquid mushroom protein hydrolysate yielded 0.77-0.92% crude protein (p>0.05). Bunashimeji mushroom protein hydrolysate was the lightest in colour, while shiitake mushroom protein hydrolysate was the darkest (p<0.05). Enoki mushroom protein hydrolysate had the highest dry matter content. There was no significant difference in the degree of hydrolysis among different mushroom protein hydrolysates (53.52-67.13%, p>0.05), with the highest yield of bunashimeji and the lowest of shiitake mushroom protein hydrolysate (p<0.05). Preference test of chicken soup with added different mushroom protein hydrolysates was performed using 58 untrained panellists to evaluate their taste-enhancing effect, compared to monosodium glutamate (MSG). Soup with MSG had the highest score for the tested attributes, while soups with bunashimeji and oyster mushroom protein hydrolysates showed higher aroma, taste, mouthfeel and overall preference scores than negative control, which contained neither MSG nor any of the hydrolysates (p<0.05). This finding suggests that bunashimeji and oyster mushroom protein hydrolysate have the potential to be used as taste enhancers in food applications.
Despite the multiple health benefits, natural flavonoid apigenin has poor aqueous solubility that restricts its delivery in foods. This study investigated the potential of spray-dried chitosan-coated liposomes prepared from scalable methods for the food industry as the delivery carriers for apigenin. Apigenin-loaded small unilamellar liposomes produced from ethanol injection had an encapsulation efficiency of 74.88 ± 5.31%. They were electrostatically stabilised via chitosan coating (0.25% w/v) and spray-dried. Spray-dried chitosan-coated apigenin liposomes (SCAL) exhibited the following powder characteristics: yield 66.62 ± 3.08%, moisture content 4.33 ± 0.56%, water activity 0.2242 ± 0.0548, particle size 10.97 ± 1.55 μm, nearly spherical morphology with wrinkles and dents under microscopic observation. Compared with the unencapsulated apigenin, SCAL demonstrated improved aqueous solubility (10.22 ± 0.18 mg/L), higher antioxidant capacity, and stability against simulated gastrointestinal digestion. The chitosan coating gave a slower in-vitro release of apigenin in SCAL (77.0 ± 6.2%) than that of uncoated apigenin liposomes (94.0 ± 5.3%) at 12 h. The apigenin release kinetics from SCAL could be represented by the Korsmeyer-Peppas model (R2 = 0.971). These findings suggest that SCAL could be a promising delivery system of apigenin for functional food applications.
Cytochrome P450s are a superfamily of heme monooxygenases which catalyze a wide range of biochemical reactions. The reactions involve the introduction of an oxygen atom into an inactivated carbon of a compound which is essential to produce an intermediate of a hydroxylated product. The diversity of chemical reactions catalyzed by cytochrome P450s has led to their increased demand in numerous industrial and biotechnology applications. A recent study showed that a gene sequence encoding a CYP was found in the genome of Bacillus lehensis G1, and this gene shared structural similarity with the bacterial vitamin D hydroxylase (Vdh) from Pseudonocardia autotrophica. The objectives of present study was to mine, for a novel CYP from a new isolate B. lehensis G1 alkaliphile and determine the biological properties and functionalities of CYP in this bacterium. Our study employed the usage of computational methods to search for the novel CYP from CYP structural databases to identify the conserved pattern, functional domain and sequence properties of the uncharacterized CYP from B. lehensis G1. A computational homology model of the protein's structure was generated and a docking analysis was performed to provide useful structural knowledge on the enzyme's possible substrate and their interaction. Sequence analysis indicated that the newly identified CYP, termed CYP107CB2, contained the fingerprint heme binding sequence motif FxxGxxxCxG at position 336-345 as well as other highly conserved motifs characteristic of cytochrome P450 proteins. Using docking studies, we identified Ser-79, Leu-81, Val-231, Val-279, Val-383, Ala-232, Thr-236 and Thr-283 as important active site residues capable of stabilizing interactions with several potential substrates, including vitamin D3, 25-hydroxyvitamin D3 and 1α-hydroxyvitamin D3, in which all substrates docked proximally to the enzyme's heme center. Biochemical analysis indicated that CYP107CB2 is a biologically active protein to produce 1α,25-dihydroxyvitamin D3 from 1α-hydroxyvitamin D3. Based on these results, we conclude that the novel CYP107CB2 identified from B. lehensis G1 is a putative vitamin D hydroxylase which is possibly capable of catalyzing the bioconversion of parental vitamin D3 to calcitriol, or related metabolic products.
The bioconversion of vitamin D3 catalyzed by cytochrome P450 (CYP) requires 25-hydroxylation and subsequent 1α-hydroxylation to produce the hormonal activated 1α,25-dihydroxyvitamin D3. Vitamin D3 25-hydroxylase catalyses the first step in the vitamin D3 biosynthetic pathway, essential in the de novo activation of vitamin D3. A CYP known as CYP107CB2 has been identified as a novel vitamin D hydroxylase in Bacillus lehensis G1. In order to deepen the understanding of this bacterial origin CYP107CB2, its detailed biological functions as well as biochemical characteristics were defined. CYP107CB2 was characterized through the absorption spectral analysis and accordingly, the enzyme was assayed for vitamin D3 hydroxylation activity. CYP-ligand characterization and catalysis optimization were conducted to increase the turnover of hydroxylated products in an NADPH-regenerating system. Results revealed that the over-expressed CYP107CB2 protein was dominantly cytosolic and the purified fraction showed a protein band at approximately 62 kDa on SDS-PAGE, indicative of CYP107CB2. Spectral analysis indicated that CYP107CB2 protein was properly folded and it was in the active form to catalyze vitamin D3 reaction at C25. HPLC and MS analysis from a reconstituted enzymatic reaction confirmed the hydroxylated products were 25-hydroxyitamin D3 and 1α,25-dihydroxyvitamin D3 when the substrates vitamin D3 and 1α-hydroxyvitamin D3 were used. Biochemical characterization shows that CYP107CB2 performed hydroxylation activity at 25 °C in pH 8 and successfully increased the production of 1α,25-dihydroxyvitamin D3 up to four fold. These findings show that CYP107CB2 has a biologically relevant vitamin D3 25-hydroxylase activity and further suggest the contribution of CYP family to the metabolism of vitamin D3.