Violaxanthin is a major xanthophyll pigment in the microalga Eustigmatos cf. polyphem, but the amount produced after propagation can vary depending upon culture conditions. In this study, the effects of cultivation time, nitrogen concentration, light intensity, and culture mode on violaxanthin production were investigated. The results showed that this microalga vigorously grew and maintained a high level of violaxanthin in the fed-batch culture, and the highest violaxanthin productivity of 1.10 ± 0.03 mg L⁻¹ d⁻¹ was obtained under low light illumination with 18 mM of initial nitrogen supply for ten days. Additionally, violaxanthin was purified from E. cf. polyphem by silica gel chromatography and preparative high-performance liquid chromatography (PHPLC), and identified with high-resolution mass spectrometry (HRMS). The antioxidant activity of the purified violaxanthin was evaluated by three tests in vitro: reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2-azobis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) radical assays. The strongest inhibition of purified violaxanthin occurred during the scavenging of ABTS⁺ radicals, with EC₅₀ of 15.25 μg mL⁻¹. In conclusion, this is the first report to investigate the effects of different culture conditions on violaxanthin accumulation in E. cf. polyphem and provide a novel source for the production of violaxanthin that can be used for food and pharmaceutical applications.

Among all organisms tested, Haematococcus pluvialis can accumulate the highest levels of natural astaxanthin. Nitrogen starvation and high irradiance promote the accumulation of starch, lipid, and astaxanthin in H. pluvialis, yet their cell division is significantly retarded. Accordingly, adaptive regulatory mechanisms are very important and necessary to optimize the cultivation conditions enabling an increase in biomass; as well as promoting astaxanthin accumulation by H. pluvialis. To clarify the intrinsic mechanism of high-level astaxanthin and biomass accumulation in the newly isolated strain, H. pluvialis JNU35, nitrogen-sufficiency and nitrogen-depletion conditions were employed. Time-resolved comparative transcriptome analysis was also conducted by crossing the two-step culture process. In the present study, we report the overall growth and physiological, biochemical, and transcriptomic characteristics of H. pluvialis JNU35 in response to nitrogen variation. From eight sampling time-points (2 days, 4 days, 8 days, 10 days, 12 days, 14 days, 16 days, and 20 days), 25,480 differentially expressed genes were found. These genes included the significantly responsive unigenes associated with photosynthesis, astaxanthin biosynthesis, and nitrogen metabolic pathways. The expressions of all key and rate-limiting genes involved in astaxanthin synthesis were significantly upregulated. The photosynthetic pathway was found to be attenuated, whereas the ferredoxin gene was upregulated, which might activate the cyclic electron-transport chain as compensation. Moreover, the expressions of genes related to nitrogen transport and assimilation were upregulated. The expressions of genes in the proteasome pathway were also upregulated. In contrast, the chloroplasts and nonessential proteins were gradually degraded, activating the specific ornithine–urea cycle pathway. These changes may promote the sustained accumulation of astaxanthin and biomass. To the best of our knowledge, this paper is the first to investigate the long-term differences of gene expression from two-step culture process in the astaxanthin producer, H. pluvialis JNU35. According to our results, β-carotene ketolase (bkt1 and bkt2) serves as the key enzyme regulating astaxanthin accumulation in H. pluvialis JNU35. The cyclic electron-transport chain and novel nitrogen metabolic process were used adaptively as the regulatory mechanism compensating for different levels of stress. The in-depth study of these metabolic pathways and related key genes can reveal the underlying relationship between cell growth and astaxanthin accumulation in H. pluvialis JNU35.

The aim of this study is to investigate the differences in the accumulation capacity of chrysolaminarin among six Tribonema species and to isolate this polysaccharide for immunomodulatory activity evaluation. The results showed that T. aequale was the most productive strain with the highest content and productivity of chrysolaminarin, which were 17.20% (% of dry weight) and 50.91 mg/L/d, respectively. Chrysolaminarin was then extracted and isolated from this alga, and its monosaccharide composition was mainly composed of a glucose (61.39%), linked by β-D-(1→3) (main chain) and β-D-(1→6) (branch chain) glycosidic bonds, with a molecular weight of less than 6 kDa. In vitro immunomodulatory assays showed that it could activate RAW264.7 cells at a certain concentration (1000 μg/mL), as evidenced by the increased phagocytic activity and upregulated mRNA expression levels of IL-1β, IL6, TNF-α and Nos2. Moreover, Western blot revealed that this polysaccharide stimulated the phosphorylation of p-65, p-38 and JNK in NF-κB and MAPK signaling pathways. Overall, these findings provide a reference for the further development and utilization of algae-based chrysolaminarin, while also offering an in-depth understanding of the immunoregulatory mechanism.

Lipophilization is a promising way to improve the bioavailability of flavonoids. However, the traditional enzymatic esterification methods are time-consuming, and present low yields and purity. Herein, a novel membrane-based lipophilization technology—bioinspired lipase immobilized membranes (BLIMs), including CAL-B@PES, CAL-B@PDA/PES and GA/CAL-B@PDA/PES— were fabricated to improve the antioxidant flavanone glycoside hesperidin lipophilization. Via reverse filtration, PDA coating and GA crosslinking, Candida antarctica lipase B (CAL-B) was stably immobilized on membrane to fabricate BLIMs. Among the three BLIMs, GA/CAL-B@PDA/PES had the greatest enzyme activity and enzyme loading, the strongest tolerance of changes in external environmental conditions (temperatures, pH, heating time, storage time and numbers of cycles) and the highest hesperidin esterification efficiency. Moreover, the optimal operating condition for GA/CAL-B@PDA/PES fabrication was the CAL-B concentration of 0.36 mg/mL, operation pressure of 2 bar, GA concentration of 5% and crosslinking time of 1 h. Afterwards, the hesperidin esterification process did not affect the micromorphology of BLIM, but clearly improved the BLIM permeability and esterified product efficiency. The present study reveals the fabrication mechanism of BLIMs and offers insights into the optimizing strategy that governs the membrane-based lipophilization technology process.