Integration analysis of ATAC-seq and RNA-seq provides insight into fatty acid biosynthesis in Schizochytrium limacinum under nitrogen limitation stress.

BACKGROUND: Schizochytrium limacinum holds significant value utilized in the industrial-scale synthesis of natural DHA. Nitrogen-limited treatment can effectively increase the content of fatty acids and DHA, but there is currently no research on chromatin accessibility during the process of transcript regulation. The objective of this research was to delve into the workings of fatty acid production in S. limacinum by examining the accessibility of promoters and profiling gene expressions. RESULTS: Results showed that differentially accessible chromatin regions (DARs)-associated genes were enriched in fatty acid metabolism, signal transduction mechanisms, and energy production. By identifying and annotating DARs-associated motifs, the study obtained 54 target transcription factor classes, including BPC, RAMOSA1, SPI1, MYC, and MYB families. Transcriptomics results revealed that several differentially expressed genes (DEGs), including SlFAD2, SlALDH, SlCAS1, SlNSDHL, and SlDGKI, are directly related to the biosynthesis of fatty acids, meanwhile, SlRPS6KA, SlCAMK1, SlMYB3R1, and SlMYB3R5 serve as transcription factors that could potentially influence the regulation of fatty acid production. In the integration analysis of DARs and ATAC-seq, 13 genes were identified, which were shared by both DEGs and DARs-associated genes, including SlCAKM, SlRP2, SlSHOC2, SlTN, SlSGK2, SlHMP, SlOGT, SlclpB, and SlDNAAF3. CONCLUSIONS: SlCAKM may act as a negative regulator of fatty acid and DHA synthesis, while SlSGK2 may act as a positive regulator, which requires further study in the future. These insights enhance our comprehension of the processes underlying fatty acid and DHA production in S. limacinum. They also supply a foundational theoretical framework and practical assistance for the development of strains rich in fatty acids and DHA.

Effects of Methanol on Carotenoids as Well as Biomass and Fatty Acid Biosynthesis in Schizochytrium limacinum B4D1.

Schizochytrium is a promising source for the production of docosahexaenoic acid and astaxanthin. The effects of different methanol concentrations on astaxanthin, biomass, and production of the lipids, squalene, and total sterol in Schizochytrium limacinum B4D1 were investigated. Astaxanthin began to accumulate when the methanol concentration reached 3.2% and peaked at 5.6% methanol, with a 2,000-fold increase over that in the control. However, under cultivation with 5.6% methanol, the biomass, lipids, squalene, and total sterol decreased to various degrees. Transcriptomic analysis was performed to explore the effects of different methanol concentrations (0%, 3.2%, and 5.6%) on the expression profile of B4D1. Three key signaling pathways were found to play important roles in regulating cell growth and metabolism under cultivation with methanol. Five central carbon metabolism-associated genes were significantly downregulated in response to 5.6% methanol and thus were expected to result in less ATP and NADPH being available for cell growth and synthesis. High methanol conditions significantly downregulated three genes involved in fatty acid and squalene/sterol precursor biosynthesis but significantly upregulated geranylgeranyl diphosphate synthase, lycopene ß-cyclase, and ß-carotene 3-hydroxylase, which are involved in astaxanthin synthesis, thus resulting in an increase in the levels of precursors and the final production of astaxanthin. Additionally, the transcriptional levels of three stress response genes were upregulated. This study investigates gene expression profiles in the astaxanthin producer Schizochytrium when grown under various methanol concentrations. These results broaden current knowledge regarding genetic expression and provide important information for promoting astaxanthin biosynthesis in SchizochytriumIMPORTANCESchizochytrium strains are usually studied as oil-producing strains, but they can also synthesize other secondary metabolites, such as astaxanthin. In this study, methanol was used as an inducer, and we explored its effects on the production of astaxanthin, a highly valuable substance in Schizochytrium Methanol induced Schizochytrium to synthesize large amounts of astaxanthin. Transcriptomic analysis was used to investigate the regulation of signaling and metabolic pathways (mainly relative gene expression) in Schizochytrium grown in the presence of various concentrations of methanol. These results contribute to the understanding of the underlying molecular mechanisms and may aid in the future optimization of Schizochytrium for astaxanthin biosynthesis.

Evaluation of Lactococcus lactis HNL12 combined with Schizochytrium limacinum algal meal in diets for humpback grouper (Cromileptes altivelis).

The humpback grouper (Cromileptes altivelis) is a commercially valuable species of the family Epinephelidae; however, its marketization suffers from slow growth speed, low survival rate, and various pathogenic diseases. Lactococcus lactis and Schizochytrium limacinum are commonly used as immunostimulants due to their health benefits for the aquatic organisms. In the present study, we assessed the effects of dietary supplementation with L. lactis HNL12 combined with S. limacinum algal meal on the growth performances, innate immune response, and disease resistance of C. altivelis against Vibrio harveyi. The results showed that fish fed with a combination diet of L. lactis and S. limacinum exhibited significantly higher final weight, percent weight gain, and specific growth rate compared with groups fed with them alone. A bacterial challenge experiment indicated that the group fed with the L. lactis combined with S. limacinum diet achieved the highest relative percent of survival value (68.63%), suggesting that L. lactis and S. limacinum significantly improved the disease resistance against V. harveyi after a 4-week feeding trial. Moreover, the respiratory burst activity of macrophages of fish fed with a L. lactis combined with S. limacinum diet was significantly higher than that of fish fed the control diet after 1, 2, and 3 weeks of feeding. The serum superoxide dismutase of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to those fed the control diet after 1 and 2 weeks of feeding, while the serum alkaline phosphatase of fish fed with a L. lactis combined with S. limacinum diet after 2 and 4 weeks was significantly increased, compared to the control group. The serum lysozyme activities of fish fed with a L. lactis combined with S. limacinum diet significantly increased compared to the control group after 2 weeks of feeding. Furthermore, transcriptome sequencing of the C. altivelis head kidney was conducted to explore the immune-regulating effects of the L. lactis combined with S. limacinum diet on C. altivelis. A total of 86,919 unigenes, annotated by at least one of the reference databases (Nr, Swiss-Prot, GO, COG, and KEGG), were assembly yielded by de novo transcriptome. In addition, 157 putative differentially expressed genes (DEGs) were identified between the L. lactis combined with S. limacinum group and the control group. For pathway enrichment, the DEGs were categorized into nine KEGG pathways, which were mainly related to infective diseases, antigen processing and presentation, digestive system, and other immune system responses. The findings of this study suggest that the L. lactis combined with S. limacinum diet can induce positive effects on the growth, immunity, and disease resistance of C. altivelis against V. harveyi. This study expands our understanding of the synergistic combinations of probiotics and prebiotics in aquaculture.

Purification and Identification of Antioxidant Peptides from Schizochytrium Limacinum Hydrolysates by Consecutive Chromatography and Electrospray Ionization-Mass Spectrometry.

Schizochytrium limacinum residue was hydrolyzed with various proteases (papain, trypsin, Flavourzyme, Protamex, and Alcalase 2.4L) to obtain antioxidative peptides. The results showed that the S. limacinum hydrolysates (SLHs) prepared with compound proteases (Protamex and Alcalase 2.4L) had the highest antioxidant activity, which was measured using methods such as 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability (IC50 = 1.28 mg/mL), hydroxyl radical scavenging ability (IC50 = 1.66 mg/mL), and reducing power (1.42 at 5.0 mg/mL). The hydrolysates were isolated and purified by ultrafiltration, gel filtration chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). Through analysis of electrospray ionization-mass spectrometer (ESI-MS/MS), the purified antioxidant peptide was identified as Pro-Tyr-Lys (406 Da). Finally, the identified peptide was synthesized for evaluating its antioxidant activity. The •OH scavenging ability and reducing power of Pro-Tyr-Lys were comparable to those of reduced L-glutathione (GSH). These results demonstrated that the antioxidant peptides from SLHs could potentially be used as effective antioxidants.

Valorification of crude glycerol for pure fractions of docosahexaenoic acid and ß-carotene production by using Schizochytrium limacinum and Blakeslea trispora.

The goal of this research is the investigation of a way to maximize the production of docosahexaenoic acid (DHA) and ß-carotene by optimizing the culture conditions of their sources, microalgae Schizochytrium limacinum and fungus Blakeslea trispora respectively, in a fermentation medium. The influencing factors in the fermentation process for producing DHA and ß-carotene have proven to be: the concentration of carbon source (different glycerol crude and pure concentrations) for both of them, and in particular temperature for DHA and pH for ß-carotene. Testing the effect of these parameters was determined: biomass, DHA and ß-carotene concentration. The highest production by S. limacinum was obtained at 25 °C, while using a quantity of 90 g/L of glycerol (crude or pure) as a carbon source. Temperature was the main factor that influenced the biosynthesis of DHA. The quantification of DHA was made by GC-MS chromatography, followed by a purification process, with the end result of DHA in pure phase. The maximum quantities for ß-carotene production were obtained with pH 7 and 60 g/L of crude glycerol. The results highlight the possibility of using crude glycerol as a low-cost substrates for growth of microalgae S. limacinum and of fungus B. trispora in order to obtain the crucial molecules: docosahexaenoic acid and ß-carotene.

Use of saline waste water from demineralization of cheese whey for cultivation of Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4.

Saline waste water from demineralization of cheese whey was used as the main component of waste saline medium (WSM) for cultivation of thraustochytrids. The suitability of WSM for cultivation of Schizochytrium limacinum PA-968 and Japonochytrium marinum AN-4 was evaluated by comparison with cultivation on nutrient medium (NM) in shake flask and fermenter cultures. Biomass productivities achieved in WSM for the thraustochytrids were comparable with those in NM for both shake flask and fermenter cultures. The maximum total lipid content (56.71% dry cell weight) and docosahexaenoic acid productivity (0.86 g/L/day) were achieved by J. marinum AN-4 grown on WSM in shake flask and fermenter cultures, respectively. A cost estimate of WSM suggests that this medium could result in lower production costs for thraustochytrid biomass and lipids and contribute to the effective reduction in saline diary process waste water.

Knockout of a PLD gene in Schizochytrium limacinum SR21 enhances docosahexaenoic acid accumulation by modulation of the phospholipid profile.

BACKGROUND: The hydrolysis and transphosphatidylation of phospholipase D (PLD) play important roles in the interconversion of phospholipids (PLs), which has been shown to profoundly impact lipid metabolism in plants. In this study, the effect of the PLD1 gene of Schizochytrium limacinum SR21 (S. limacinum SR21) on lipid metabolism was investigated. RESULTS: PLD1 knockout had little impact on cell growth and lipid production, but it significantly improved the percentage of polyunsaturated fatty acids in lipids, of which docosahexaenoic acid (DHA) content increased by 13.3% compared to the wild-type strain. Phospholipomics and real-time quantitative PCR analysis revealed the knockout of PLD1 reduced the interexchange and increased de novo synthesis of PLs, which altered the composition of PLs, accompanied by a final decrease in phosphatidylcholine (PC) and an increase in phosphatidylinositol, lysophosphatidylcholine, and phosphatidic acid levels. PLD1 knockout also increased DHA content in triglycerides (TAGs) and decreased it in PLs. CONCLUSIONS: These results indicate that PLD1 mainly performs the transphosphatidylation activity in S. limacinum SR21, and its knockout promotes the migration of DHA from PLs to TAGs, which is conducive to DHA accumulation and storage in TAGs via an acyl CoA-independent pathway. This study provides a novel approach for identifying the mechanism of DHA accumulation and metabolic regulation strategies for DHA production in S. limacinum SR21.

Evaluation of Growth Performance and Environmental Impact of Hermetia illucens Larvae Reared on Coffee Silverskins Enriched with Schizochytrium limacinum or Isochrysis galbana Microalgae.

Hermetia illucens is a promising insect due to its ability to convert low-value substrates as food chain by-products into highly nutritious feed. Its feeding and nutrition are important issues. The aim of this work was to investigate the effect of different substrates consisting of coffee silverskin, a by-product of the roasting process, enriched with different inclusions of microalgae (5%, 10%, 20%, and 25%), Schizochytrium limacinum, and Isochrysis galbana, combined with the assessment of environmental sustainability by LCA. In general, the addition of microalgae led to an increase in larval growth performance due to the higher content of protein and lipids, although S. limacinum showed the best results with respect to larvae fed with coffee silverskin enriched with I. galbana. A higher prepupal weight was observed in larvae fed with 10%, 20%, and 25% S. limacinum; shorter development times in larvae fed with 25% of both S. limacinum and I. galbana; and a higher growth rate in larvae fed with 25% S. limacinum. The 10% S. limacinum inclusion was only slightly different from the higher inclusions. Furthermore, 10% of S. limacinum achieved the best waste reduction index. The greater the inclusion of microalgae, the greater the environmental impact of larval production. Therefore, the addition of 10% S. limacinum appears to be the best compromise for larval rearing, especially considering that a higher inclusion of microalgae did not yield additional benefits in terms of the nutritional value of H. illucens prepupae.

Changes in Chemical Composition and Fatty Acid Profile of Milk and Cheese and Sensory Profile of Milk via Supplementation of Goats' Diet with Marine Algae.

The aim of the present study was to assess the effects of the low level of Schizochytrium limacinum marine algae (daily 5 g per animal) on the milk, cheese, and whey composition; fatty acid profile of milk and cheese; and the sensory profile of goat milk using an e-nose device. Thirty Alpine goats were randomly divided into two groups: the control group (C, n = 15)-fed grass with daily 600 g concentrate and the experimental group (MA, n = 15) who received the same forage and concentrate supplemented with 5 g/head/day marine algae. Animals were kept indoors and the investigation period lasted 52 days, including the first six weeks as the period of adaptation and the last 10 days as the treatment period. During the adaptation period, bulk milk samples from each group were collected once a week (0, 7, 14, 21, 28, 35, and 42 d), while during the treatment period (10 days), bulk milk samples from each group were taken every day, and cheese samples were processed from bulk milk each day from both groups. Marine algae supplementation had no negative effect on milk composition. In contrast, the marine algae inclusion significantly elevated the fat and protein content of whey and the protein content of cheese, as well as the recovery of fat and protein in the curd, while increasing the cheeses' moisture content on a fat-free basis. The marine algae supplementation significantly increased the docosahexaenoic acid (DHA) and the rumenic acid (CLA c9t11) concentrations and decreased the n-6/n-3 ratio in the milk and cheese. There were no significant differences between the C and the MA group with regard to the sensory profiles of the milk. It can be concluded that the milk obtained from goats given daily supplementation of 5g of MA has a fatty acid profile more beneficial to human health, without any negative effects on the milk's aromatic components.

Optimization of Lipid Production by Schizochytrium limacinum Biomass Modified with Ethyl Methane Sulfonate and Grown on Waste Glycerol.

One of the most promising avenues of biofuel research relates to using waste as a starting feedstock to produce liquid or gaseous energy carriers. The global production of waste glycerol by the refinery industry is rising year after year. The aim of the present study was to examine the effect of ethyl methane sulfonate (EMS) on the growth rates and intracellular lipid accumulation in heterotrophically-cultured Schizochytrium limacinum microalgae, grown on waste glycerol as the carbon source. The strain S. limacinum E20, produced by incubating a reference strain in EMS for 20 min, was found to perform the best in terms of producing biomass (0.054 gDW/dm3·h) and accumulating intracellular bio-oil (0.021 g/dm3·h). The selected parameters proved to be optimal for S. limacinum E20 biomass growth at the following values: temperature 27.3 °C, glycerol level 249.0 g/dm3, oxygen in the culture 26%, and yeast extract concentration 45.0 g/dm3. In turn, the optimal values for lipid production in an S. limacinum E20 culture were: temperature 24.2 °C, glycerol level 223.0 g/dm3, oxygen in the culture 10%, and yeast extract concentration 10.0 g/dm3. As the process conditions are different for biomass growth and for intracellular lipid accumulation, it is recommended to use a two-step culture process, which resulted in a lipid synthesis rate of 0.41 g/dm3·h.

Effect of Marine Algae Supplementation on Somatic Cell Count, Prevalence of Udder Pathogens, and Fatty Acid Profile of Dairy Goats' Milk.

The aim of this study was to evaluate the effect of the Schizochytrium limacinum marine algae on the milk composition and fatty acid profile, somatic cell count, and prevalence of pathogen bacteria in the raw milk of multiparous Alpine goats. Twenty-eight dairy goats were randomly allocated to two groups: control group (C)-fed with 1500 g alfalfa hay and 600 g concentrate; experimental group (MA)-received the same forages and concentrate supplemented with 10 g/head/day marine algae. The goats were housed indoors, while the experiment lasted five weeks, and the milk samples were taken every week. Marine algae feeding had no negative effect on milk composition. The marine algae inclusion significantly decreased the milk somatic cell count and the presence of udder pathogens in the MA group. Mean somatic cell count and presence of udder pathogens were 5.73 log cells/mL and 31%, respectively, in the C group, while these values were 5.34 log cells/mL and 10%, respectively, in the MA group. The marine algae supplementation significantly increased DHA and rumenic acid concentration in the milk of the MA group (0.32 and 0.99 g/100 g of fatty acids, respectively) compared to the C group (0.04 and 0.65 g/100 g of fatty acids, respectively). It can be concluded that a diet supplemented with marine algae significantly improves the udder health of goats and the concentrations of health-promoting fatty acids in milk.

Function of ORFC of the polyketide synthase gene cluster on fatty acid accumulation in Schizochytrium limacinum SR21.

BACKGROUND: As a potential source of polyunsaturated fatty acids (PUFA), Schizochytrium sp. has been widely used in industry for PUFA production. Polyketide synthase (PKS) cluster is supposed to be the primary way of PUFA synthesis in Schizochytrium sp. As one of three open reading frames (ORF) in the PKS cluster, ORFC plays an essential role in fatty acid biosynthesis. However, the function of domains in ORFC in the fatty acid synthesis of Schizochytrium sp. remained unclear. RESULTS: In this study, heterologous expression and overexpression were carried out to study the role of ORFC and its domains in fatty acid accumulation. Firstly, ORFC was heterologously expressed in yeast which increased the PUFA content significantly. Then, the dehydratase (DH) and enoyl reductase (ER) domains located on ORFC were overexpressed in Schizochytrium limacinum SR21, respectively. Fatty acids profile analysis showed that the contents of PUFA and saturated fatty acid were increased in the DH and ER overexpression strains, respectively. This indicated that the DH and ER domains played distinct roles in lipid accumulation. Metabolic and transcriptomic analysis revealed that the pentose phosphate pathway and triacylglycerol biosynthesis were enhanced, while the tricarboxylic acid cycle and fatty acids oxidation were weakened in DH-overexpression strain. However, the opposite effect was found in the ER-overexpression strain. CONCLUSION: Therefore, ORFC was required for the biosynthesis of fatty acid. The DH domain played a crucial role in PUFA synthesis, whereas the ER domain might be related to saturated fatty acids (SFA) synthesis in Schizochytrium limacinum SR21. This research explored the role of ORFC in the PKS gene cluster in Schizochytrium limacinum and provided potential genetic modification strategies for improving lipid production and regulating PUFA and SFA content.

Microbial dynamics in rearing trials of Hermetia illucens larvae fed coffee silverskin and microalgae.

In the present study, Hermetia illucens larvae were reared on a main rearing substrate composed of a coffee roasting byproduct (coffee silverskin, Cs) enriched with microalgae (Schizochytrium limacinum or Isochrysis galbana) at various substitution levels. The microbial diversity of the rearing substrates, larvae, and frass (excrement from the larvae mixed with the substrate residue) were studied by the combination of microbial culturing on various growth media and metataxonomic analysis (Illumina sequencing). High counts of total mesophilic aerobes, bacterial spores, presumptive lactic acid bacteria, coagulase-positive cocci, and eumycetes were detected. Enterobacteriaceae counts were low in the rearing diets, whereas higher counts of this microbial family were observed in the larvae and frass. The microbiota of the rearing substrates was characterized by the presence of lactic acid bacteria, including the genera Lactobacillus, Leuconostoc and Weissella. The microbiota of the H. illucens larvae fed Cs was characterized by the dominance of Paenibacillus. H. illucens fed diets containing I. galbana were characterized by the presence of Enterococcus, Lysinibacillus, Morganella, and Paenibacillus, depending on the algae inclusion level, while H. illucens fed diets containing S. limacinum were characterized by high relative abundances of Brevundimonas, Enterococcus, Paracoccus, and Paenibacillus, depending on the algae inclusion level. Brevundimonas and Alcaligenes dominated in the frass from larvae fed I. galbana; the predominance of Brevundimonas was also observed in the frass from larvae fed Schyzochitrium-enriched diets. Based on the results of the present study, an effect of algae nutrient bioactive substances (e.g. polysaccharides, high-unsaturated fatty acids, taurine, carotenoids) on the relative abundance of some of the bacterial taxa detected in larvae may be hypothesized, thus opening new intriguing perspectives for the control of the entomopathogenic species and foodborne human pathogens potentially occurring in edible insects. Further studies are needed to support this hypothesis. Finally, new information on the microbial diversity occurring in insect frass was also obtained.

Safety of oil from Schizochytrium limacinum (strain FCC-3204) for use in infant and follow-on formula as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of Schizochytrium sp. oil as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Schizochytrium sp. is a single-cell microalga. The strain FCC- 3204, used by the applicant (Fermentalg), belongs to the species Schizochytrium limacinum. The NF, an oil rich in docosahexaenoic acid (DHA), is obtained from microalgae after enzymatic lysis. The applicant proposed to use the NF in infant formulae (IF) and follow-on formulae (FOF). The use level defined by the applicant was derived from Regulation (EU) 2016/127, which states the mandatory addition of DHA to IF and FOF at the level of 20-50 mg/100 kcal. The intake of DHA resulting from the use of the NF in IF and FOF is not expected to pose safety concerns. S. limacinum was attributed the qualified presumption of safety (QPS) status with the qualification 'for production purposes only'. Data provided by the applicant demonstrated the absence of viable cells in the NF. No toxicological studies were performed with the NF. However, based on the available toxicological data on oils derived from Schizochytrium sp., the QPS status of the source of the NF, the production process, the composition of the NF and the absence of viable cells in the NF, the Panel considers there are no concerns with regard to toxicity of the NF. The Panel concludes that the NF is safe under the proposed conditions of use.

Safety of oil from Schizochytrium limacinum (strain FCC-3204) for use in food supplements as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of Schizochytrium sp. oil as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Schizochytrium sp. is a single-cell microalga. The strain FCC-3204, used by the applicant (Fermentalg), belongs to the species Schizochytrium limacinum. The NF, an oil rich in docosahexaenoic acid (DHA), is obtained from microalgae after enzymatic lysis. The applicant proposed to increase the use level of the NF as a food supplement, from 250 mg DHA/day (currently authorised for the general population, excluding pregnant and lactating women) to 3 g DHA/day for adults, excluding pregnant and lactating women. S. limacinum was attributed the qualified presumption of safety (QPS) status with the qualification 'for production purposes only'. Data provided by the applicant demonstrated the absence of viable cells in the NF. No toxicological studies were performed with the NF. However, based on the available toxicological data on oils derived from Schizochytrium sp., the QPS status of the source of the NF, the production process, the composition of the NF and the absence of viable cells in the NF, the Panel considers there are no concerns with regard to toxicity of the NF. The Panel considers that the data provided by the applicant are not sufficient to conclude on the safety of the NF at the proposed uses (3 g DHA/day as a food supplement) in adults. However, in 2012, the Panel concluded that supplemental intakes of DHA alone up to about 1 g/day do not raise safety concerns for the general population. The Panel concludes that the NF is safe for the use in food supplements at the maximum intake level of 1 g DHA/day for the target population (adults, excluding pregnant and lactating women).

Schizochytrium limacinum Supplementation in a Low Fish-Meal Diet Improved Immune Response and Intestinal Health of Juvenile Penaeus monodon.

The aim of the present experiment was to evaluate the effects of Schizochytrium limacinum supplementation on the immune response, gut microbiota, and health of Penaeus monodon fed a low fish-meal (FM) diet. A diet containing 25% FM was used as a control (Diet A), and three other diets were formulated to contain 15% FM and supplemented with 0, 0.75, and 1.5% S. limacinum (Diet B, C, and D, respectively). The experiment was carried out in quadruplicates (30 shrimp per replicate, average weight 1.01 ± 0.01 g), and the shrimps were fed the test diets to apparent satiation three times daily for 8 weeks. Shrimp fed diet B and D showed lower weight gain than those fed diet A. Supplementation of 0.75% S. limacinum enhanced expression of antioxidative genes (superoxide dismutase and catalase) and immune-response-related genes in hepatopancreas but could not affect the gene expression of immune deficiency in hepatopancreas and Tube in the intestine. A low FM diet induced endoplasmic reticulum swelling of the intestinal epithelial cells, which was alleviated by S. limacinum supplementation. Ultra-performance liquid chromatography coupled with quadrupole time of flight mass spectrometry was employed to analyze the changes of hemolymph metabolomics, 49 significantly different metabolites were identified, and lysoPCs, deoxyinosine, inosine, and highly unsaturated fatty acids were lower in fish fed with low FM diets. Intestinal microbial diversity was lower in shrimp fed Diet B than those fed the control diet. Dietary supplementation of 0.75% S. limacinum increased intestinal microbial diversity of shrimp and decreased the ratio of pathogenic bacterium (Thalassotalea and Tenacibaculum). These results indicated that supplementing S. limacinum into a low FM diet improves the growth performance, immune response, and intestinal health of P. monodon. The optimum inclusion level of seems to be 0.75% of diet.

Genome and Transcriptome Analyses Provide Insight Into the Omega-3 Long-Chain Polyunsaturated Fatty Acids Biosynthesis of Schizochytrium limacinum SR21.

Schizochytrium sp. is the best natural resource for omega-3 long-chain polyunsaturated fatty acids. We report a high-quality genome sequence of Schizochytrium limacinum SR21, which has a 63 Mb genome size, with a contig N50 of 2.67 Mb and 6,838 protein-coding genes. Phylogenomic and comparative genomic analyses revealed that DHA-producing Schizochytrium and Aurantiochytrium strains were highly similar and possessed similar genes. Analysis of the fatty acid synthase (FAS) for LC-PUFAs production results in the annotation of all genes in map00062 and map01212. A gene cluster and 10 ORFs related to PKS pathway were found in the genome. 1,402 differentially expressed genes (DEGs) of the treated groups (0.5 g/L yeast extract) were identified by comparing with the control groups (1.0 g/L yeast extract) at 36 h. A weighted gene coexpression network analysis revealed that 2 of 7 modules correlated highly with the fatty acid and DHA contents. The DEGs and transcription factors were significantly correlated with fatty acid biosynthesis, including MYB, Zinc Finger and ACOX. The results showed that these hub genes are regulated by genes involved in fatty acid biosynthesis pathways. The results providing an important reference for further research on promoting fatty acid and DHA accumulation in S. limacinum SR21.

High-Density pH-Auxostat Fed-Batch Culture of Schizochytrium limacinum SR21 with Acetic Acid as a Carbon Source.

Schizochytrium limacinum SR21 is an important strain for industrial production of docosahexaenoic acid (DHA), which is an important omega-3 fatty acid used in the nutraceutical and food industry. However, the high cost of carbon sources has limited its further application in the market with much larger volume, such as animal feed for aquaculture, poultry, and livestock. To seek low-cost carbon source, acetic acid is tested in the present study. The effect of different factors, including initial carbon source concentration, pH, aeration rate, and nitrogen sources, on biomass, lipid, and DHA production were tested. With optimized culture conditions, the biomass concentration of 146 g/L, total fatty acids (TFAs) of 82.3 g/L, and DHA content of 23.0 g/L were achieved with a pH-auxostat fed-batch cultivation. These results suggested that acetic acid is a promising feedstock for the low-cost production of DHA. Graphical Abstract.

Feeding to Produce n-3 Fatty Acid-enriched Table Eggs.

This study aimed to modify the feed mixtures of laying hens to enrich the consumer eggs with n-3 polyunsaturated fatty acids (PUFA): α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). One hundred and twenty Tetra-SL laying hens used in the study were divided into three groups of 40 laying hens arranged in five repetitions: C, control with 5% soybean oil; E1, 0.5% fish oil + 0.5% microalgae Schizochytrium limacinum; and E2, 0.75% fish oil + 0.75% microalgae. The composition of the mixtures was balanced at the level of 17.5% raw protein and 11.81 MJ/kg metabolic energy (ME). Feed and water were provided ad libitum, and the experiment lasted for 21 days. In this study, the different physical and chemical properties of eggs, the fatty acid profile and lipid oxidation of fat in egg yolks were analyzed. The results of the study showed that the weight of the egg yolk and that of the shell depended on the feeding treatments (P=0.014 and P<0.001), and the weight of eggs and basic parts, as well as the thickness of the shell depended on the storage duration (P<0.001). The storage time affected the pH of egg yolks and albumen and the reduction in Haugh units and albumen height (P<0.001). Significant differences were observed in the content of ALA, DHA, ∑n-3 PUFA (mg/100 g) and the n-6/n-3 PUFA ratio between the C and E1/E2 egg groups (P<0.001). The results of the study indicate that it is sufficient to use a lower level of fish oil and the microalgae Schizochytrium limacinum in hens' feed to achieve a satisfactory increase in n-3 PUFA in eggs, while maintaining optimal values of egg quality and freshness indicators.

Safety of Schizochytrium sp. oil as a novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the safety of Schizochytrium sp. oil as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Schizochytrium sp. is a single-cell microalga. The strain WZU477, used by the applicant (Progress Biotech bv), was found to belong to the species Schizochytrium limacinum and was obtained in a marine environment from rotted mangrove forest leaves. The NF, an oil rich in docosahexaenoic acid (DHA), is isolated from the microalgae by mechanical extraction. The applicant proposed to use the NF in infant formulae (IF) and follow-on formulae (FOF). The use level defined by the applicant was derived from Regulation (EU) 2016/127, which states the mandatory addition of DHA to IF and FOF at the level of 20-50 mg/100 kcal. The intake of DHA resulting from the use of the NF in IF and FOF is not expected to pose safety concerns. The composition of the NF indicates the absence of marine biotoxins in the NF. Furthermore, Schizochytrium limacinum was attributed the qualified presumption of safety (QPS) status with the qualification 'for production purposes only'. Based on the information provided, the microalga is not expected to survive the manufacturing process. Toxicological tests conducted with the NF were not performed. However, based on the available toxicological data on various forms of oils derived from Schizochytrium sp., the QPS status of the source of the NF, the production process and the composition of the NF, the Panel considers there are no concerns with regard to toxicity of the NF. The Panel concludes that the NF is safe under the proposed conditions of use.