Identification of vitamin B12 and pseudovitamin B12 from various edible shellfish using liquid chromatography–electrospray ionization/tandem mass spectrometry

In this study, the vitamin B₁₂ contents were analyzed in the edible portions of various shellfish (bivalves and snails). High vitamin B₁₂ contents (30.5–53.3 μg/100 g wet weight) were detected in mussels, surf clams, bloody clams, and freshwater clams. However, scallops and abalone had extremely low vitamin B₁₂ contents (0.1–1.1 μg/100 g wet weight) which was attributed to only the muscle portions being edible. These results suggest that high levels of vitamin B₁₂ are accumulated in the viscera of shellfish. Vitamin B₁₂ levels were also significantly higher in bivalves than in snails. The corrinoid compounds purified from all bivalves were identified as “true” vitamin B₁₂ using liquid chromatography–electrospray ionization/tandem mass spectrometry. In edible snails, abalone, and pond snails, however, both vitamin B₁₂ and pseudovitamin B₁₂ (an inactive corrinoid) were observed to be the major and minor corrinoid compounds, respectively. Based on these results, we conclude that the whole bodies of these edible bivalves are excellent sources of vitamin B₁₂ for humans.     

Characterization of vitamin B12 compounds from commercially available fish roe products

Although fish roes (or the egg-laden ovary) are seafood products with high nutritional value and are considered abundant in vitamins including vitamin B₁₂, nevertheless, the detailed properties of vitamin B₁₂ have not been fully elucidated in fish roe products. Vitamin B₁₂ content was determined using C18-reversed-phase high-performance liquid chromatography after purification of samples from immunoaffinity columns. Salmon egg-laden ovary products (sujiko), salmon roe products (ikura), dried mullet roe products (bottarga), and white sturgeon roe products (caviar) were found to contain substantial levels of vitamin B₁₂ (more than approximately 15 µg/100 g wet weight). Interestingly, high levels of vitamin B₁₂ per 100 g wet weight have been measured in pollack roe products (tarako) and flying fish roe products (tobiko). Liquid chromatography–electrospray-ionization/tandem mass spectrometry analysis revealed that vitamin B₁₂ was the predominant corrinoid in the evaluated fish roe products examined, while no inactive corrinoid compounds were identified. These results suggest that commercially available fish roe products could be an important source of vitamin B₁₂ for use as a dietary product for humans.

     

Characterization of vitamin B<Subscript>12</Subscript> compounds from marine foods

Vitamin B₁₂ is synthesized by only certain bacteria and archaea but not by animals or plants. In marine environments, bacterial vitamin B₁₂ is transferred and concentrated into fish and shellfish bodies by plankton in the marine food chain. Moreover, marine macrophytic red algae, Porphyra spp. specifically contain substantial amounts of vitamin B₁₂, due to microbial interaction. Although some meats or viscera of edible fish and shellfish are excellent sources of biologically active vitamin B₁₂, an inactive corrinoid, pseudovitamin B₁₂, was found in some edible shellfish using liquid chromatography/electrospray ionization–tandem mass spectrometry. To prevent elderly people from developing vitamin B₁₂ deficiency due to food protein-bound vitamin B₁₂ malabsorption, we present a survey of marine foods containing free vitamin B₁₂. The results of our study suggest that bonito and clam extracts (or soup stocks), which contain considerable amounts of free vitamin B₁₂ are useful not only as seasonings and flavorings but also as excellent sources of free vitamin B₁₂.     

Carbon dioxide fixation by the seaweed Gracilaria lemaneiformis in integrated multi-trophic aquaculture with the scallop Chlamys farreri in Sanggou Bay, China

The red alga Gracilaria lemaneiformis was cultivated with the scallop Chlamys farreri in an integrated multi-trophic aquaculture (IMTA) system for 42 h at Sanggou Bay, located in north China. Variation in inorganic carbon in the IMTA system was determined. The experiment included three treatments each with three replicates and three scallop monoculture systems as controls. Scallop density (399.1 ± 7.85 g per microcosm) remained the same in all treatments while seaweed density differed. The seaweed density was set at three levels (treatments 1, 2, 3) with thallus wet weights of 125.3 ± 4.72 g, 252.3 ± 7.50 g, and 378.7 ± 6.51 g per microcosm, respectively. This produced bivalve to seaweed wet weight ratios of 1:0.31, 1:0.63, and 1:0.96 for treatments 1, 2, and 3, respectively. In control groups, continuous dissolution of carbon dioxide (CO₂) produced by scallops into the seawater not only caused an ongoing increase in partial pressure of CO₂ (pCO₂), 5.5 times higher than that of natural seawater, but also acidified seawater by 0.8 units after 42 h of culture. However, in all seaweed-scallop groups, the higher the algal density, the more CO₂ was absorbed; pCO₂ was lowest in treatment 3. The results suggest that a ratio of bivalve to seaweed less than 1:0.96 may produce an even stronger CO₂ sink. Overall, the integrated culture of seaweed and scallop could provide an efficient and environmentally friendly means to reduce CO₂ emissions from bivalve mariculture.     

Effect of seaweed epibiotic bacterium Streptomyces violaceoruber SCH-09 on marine fouling organisms

Seaweed-associated bacteria play a role in helping to protect host organisms from biofouling by producing anti-fouling compounds. In a first step to develop such anti-fouling compounds, we isolated epibiotic bacteria from seaweed and subsequently screen culture extracts for anti-fouling activities. The active epibiotic bacterium studied was isolated from Undaria pinnatifida and identified as Streptomyces violaceoruber based on 16S rDNA sequence analysis. Two active compounds were isolated from the culture extracts by silica gel chromatography and high-performance liquid chromatography, and their structures were analyzed by nuclear magnetic resonance and high-resolution electron ionization mass spectrometry. The compounds were identified as furanone derivatives: 3-octa-1′,3′-dienyl-4-methylfuran-2(5H)-one and 3-octa-1′-enyl-4-methylfuran-2(5H)-one, respectively. These compounds showed anti-fouling activities against the following fouling organisms: zoospores of Ulva pertusa, the diatom Navicula annexa, and the mussel Mytilus edulis, with an EC₅₀ (minimum concentration for 50 % spore inhibition) range of 0.02–0.1 μg/ml. In the acute toxicity tests on juvenile rockfish Shebastes shlegelli, brine shrimp Artemia salina, and microalga Tetraselmis suecica, the two compounds showed LC₅₀ (concentration lethal to 50 % of the spores) ranges of 13.9–118.9 and 14.8–81.9 μg/ml, respectively.     

Effects of the supplementation of vitamin D<Subscript>3</Subscript> on the growth and vitamin D metabolites in juvenile Siberian sturgeon (<Emphasis Type="Italic">Acipenser baerii</Emphasis>)

The objective of this study was to evaluate the effects of the supplementation of vitamin D₃ on the growth, vitamin D metabolites, and osteocalcin secretion in juvenile Siberian sturgeon (Acipenser baerii). A 90-day growth trial was conducted with juvenile Siberian sturgeon (initial body weight 3.47 ± 0.14 g) fed seven isonitrogenous and isoenergetic practical diets (45% CP and 13% lipid) containing 60 (basal diet), 240, 450, 880, 1670, 3300, or 1.0 × 10⁵ IU/kg feed (D60~D 1.0 × 10⁵) vitamin D₃. The results showed that weight gain and specific growth rate increased as the dietary vitamin D₃ levels increased from 450 to 3300 IU/kg (P < 0.05). The fish fed with D1670 and D3300 diets had higher crude lipid and ash levels than the fish fed the D60 diet (P < 0.05). The fish fed D880, D1670, or D3300 diets had higher 25-OH-D₃ and 1,25-(OH)₂-D₃ levels than the fish fed the D60 diet (P < 0.05). The fish fed D880, D1670, D3300, or D1.0 × 10⁵ diets had higher osteocalcin levels than the fish fed the D60 diet (P < 0.05). Based on the broken line method analysis of weight gain and osteocalcin, the dietary vitamin D₃ requirement of juvenile Siberian sturgeon was estimated to be 1683.30 and 1403.27 IU/kg per diet, respectively.     

Water quality and growth of Pacific white shrimp Litopenaeus vannamei (Boone) in co-culture with green seaweed Ulva lactuca (Linaeus) in intensive system

An indoor trial was conducted for 28 days to evaluate the effects and interactions of biofloc and seaweed Ulva lactuca in water quality and growth of Pacific white shrimp Litopenaeus vannamei in intensive system. L. vannamei (4.54 ± 0.09 g) were stocked in experimental tanks at a density 132 shrimp m^?2 (566 shrimp m^?3) and the U. lactuca was stocked at a density 0.46 kg m^?2 (2.0 kg m^?3). Biofloc with seaweed (BF-S) significantly reduced (P < 0.05) total ammonia nitrogen (TAN) by 25.9 %, nitrite–nitrogen (NO₂–N) by 72.8 %, phosphate (PO ₄ ³ -P) by 24.6 %, and total suspended solids by 12.9 % in the water and significantly increased (P < 0.05) settleable solids by 34.2 % and final weight of shrimp by 6.9 % as compared to biofloc without seaweed. The BF-S can contribute by reducing nitrogen compounds (TAN and NO₂–N), phosphate (PO ₄ ³ -P), and total suspended solids in water and increased final weight of shrimp.     

The effects of dietary vitamin C on mucosal immune responses and growth performance in Caspian roach (Rutilus rutilus caspicus) fry

This study was conducted to investigate the effects of different levels of dietary vitamin C on some skin mucus immune parameters, mucus antimicrobial activity and growth performance of Caspian roach (Rutilus rutilus caspicus) fry. Six hundred sixty Caspian roach (1.4 ± 0.02 g) fry were allocated to 12 tanks (55 fish per tank), and triplicate groups were fed diets containing 0, 1,000, 1,500 and 2,000 mg kg^?1 vitamin C for 60 days. At the end of the trial, the epidermal mucus protein level, alkaline phosphatase and antimicrobial activity against two gram-positive bacteria (Streptococcus faecium and Micrococcus luteus) and gram-negative bacteria (Escherichia coli and Serratia marcescens) as well as growth performance were measured. The results demonstrated that feeding on vitamin C significantly elevated skin mucus alkaline phosphatase and protein levels compared to the control group (P < 0.05). However, lysozyme activity was undetectable in both the vitamin C-fed roach fry and the control group. Skin mucus antimicrobial activity was increased following vitamin C administration, and the bacterial growth inhibition zones were significantly elevated in vitamin C-fed roach (P < 0.05). Similar results were obtained in case of the minimum inhibitory concentration of skin mucus. Also fish fed the control diet had a significantly lower weight gain, specific growth rate and condition factor compared to the other treatments (P < 0.05). These results revealed that dietary vitamin C beneficially affects the skin mucus immune parameters and growth performance of Caspian roach fry.     

Ammonia and nitrite toxicity to false clownfish <Emphasis Type="Italic">Amphiprion ocellaris</Emphasis>

False clownfish, Amphiprion ocellaris, is one of the most commercialized fish species in the world, highly produced to supply the aquarium market. The high stocking densities used to maximize fish production can increase ammonia and nitrite to toxic levels. In this study, A. ocellaris juveniles (1.20 ± 0.34 g) were exposed to six concentrations of ammonia ranged from 0.23 to 1.63 mg/L NH₃-N and eight concentrations of nitrite (26.3–202.2 mg/L NO₂ ^?-N). The LC₅₀- 24, LC₅₀-48, LC₅₀-72 and LC₅₀-96 h were estimated to be 1.06, 0.83, 0.75 and 0.75 mg/L for NH₃-N and 188.3, 151.01, 124.1 and 108.8 mg/L for NO₂ ^?-N. Analysis of gill lesions caused by sublethal concentrations of these nitrogenous compounds showed that both nitrogenous compounds induced tissue lesions such as hyperplasia of epithelium cells, hypertrophy of chloride cells and lamellar lifting to all concentrations tested. However, histopathological alterations were more conspicuous accordingly the increase of ammonia or nitrite in fish exposed to 0.57 mg/L NH₃-N or 100 mg/L NO₂ ^?-N. Based on our results, we recommend to avoid concentrations higher than 0.57 mg/L of NH₃-N and 25 mg/L of NO₂-N in water.     

Evaluation of the pigmentation stages and body proportions from the glass eel to yellow eel in Anguilla japonica

The pigmentation stages of Anguilla japonica were classified from the glass eel to yellow eel stage using 412 wild-caught eels from Hamana Lake and detailed developmental observations of 10 laboratory-reared eels. The sequential appearance of pigment was similar to that in A. anguilla, except for delayed pigment formation on the nerve cord of A. japonica. The general classification of pigmentation stages (V_A–VI_B) in A. anguilla was applicable to A. japonica, but the V_B stage could be separated into two stages (V_B1, V_B2) in A. japonica. The completion of guanine deposition on the intra-abdominal membrane may be a trait to discriminate the yellow eel stage (VII) from the VI_B stage. In natural conditions, wild-caught 0-age eels (n = 3,298) did not increase in size and retained a slender body form until VI_A4, and then became thicker during VI_B. The transition between body forms and growth may correspond to their ecological change from using passive transport in glass eels with tidal flow into rivers, to their settlement and further movements of elvers upstream. Considering this behavioral transition and pigmentation progression, it is proposed that the terms "glass eel" and "elver" could be defined as stages V_A–VI_A4 and VI_B, respectively.     

Effects of heating conditions on fatty acids and volatile compounds in foot muscle of abalone Haliotis discus hannai Ino

Abalone Haliotis discus hannai Ino is a popular delicacy consumed as a luxury food owing to its unique flavor and texture. In this study, we investigated the effects of heating conditions on the fatty acids and volatile compounds in its foot muscle based on gas chromatography–mass spectrometry (GC–MS) and solid-phase microextraction (SPME)–GC–MS. The contents of fatty acids significantly decreased after heating at 80 °C for 2 h. In total, 52 volatile compounds, including aldehydes, aromatic compounds, alkanes, alcohols, ketones, and furans, were detected in the heated samples. Principal component analysis revealed an interaction between heating temperature and time. Heating at 80 °C for 0.5–2 h generated higher contents of volatile compounds. In particular, the contents of hexanal, heptanal, octanal, nonanal, and undecanal mainly derived from autoxidation of fatty acids during heating increased at least fourfold.     

Short communication: Acute toxicity of hydrogen peroxide in juvenile white shrimp Litopenaeus vannamei reared in biofloc technology systems

Biofloc technology (BFT) has been used to rear white shrimp, Litopenaeus vannamei. In this culturing system, the absence of aeration causes a rapid drop in dissolved oxygen levels, and hydrogen peroxide (H₂O₂) can be used as an emergency source of oxygen. This study aimed to determine the lethal concentration and safe level of H₂O₂ applied as a source of oxygen for juvenile white shrimp L. vannamei in a BFT system. Juveniles (1.39 ± 0.37 g) were exposed for 2 h to different concentrations of H₂O₂ [29 (100), 58 (200), 116 (400), 174 (600), 232 (800), 290 (1,000) and 348 (1,200) μL H₂O₂/L (ppm H₂O₂-29 %/L)] in addition to a control group without addition of H₂O₂, and the survival rates were monitored for 96 h. The LC₅₀ values and 95 % confidence intervals at 24, 48, 72 and 96 h were 235.5 (207–268), 199.1 (172–229), 171.1 (146–198) and 143.3 (120–170) μL H₂O₂/L, respectively. The safe level was 14.3 μL H₂O₂/L, and the highest concentration with survival rates similar to the control group (NOAEC) was 29 μL H₂O₂/L. In these concentrations, H₂O₂ can be used as a safe source of oxygen for L. vannamei reared in BFT systems.     

Influence of dietary vitamin B6 on tissue vitamin B6 contents and immunity in Atlantic salmon, Salmo salar L.

Atlantic salmon, Salmo salar L., (14 g) were fed a practical fish-meal-based diet supplemented with 0. 10, 20. 40, 80 and 160 mg pyridoxine (PN) per kg feed for 20 weeks. Tissue vitamin B₆ contents were significantly reduced in fish fed the non-supplemented diet compared with fish fed PN-supplemented diets. Serum haemolytic complement activity and head kidney lysozyme activity, and the specific antibody response following immunization with Vibrio salmonicida. were not influenced by the dietary regimes. Challenge with Aeromonas salmonicida showed that increasing the dietary levels of vitamin B₆ did not improve the resistance to furunculosis. Growth, mortality and haematology were not affected by supplementing a practical diet with vitamin B₆ In conclusion, feed levels of vitamin B₆ higher than the minimum dietary requirement did not enhance immune functions and disease resistance in Atlantic salmon.     

Dietary Aloe vera components’ effects on cholesterol lowering and estrogenic responses in juvenile goldfish, Carassius auratus

Aloes are now considered a very interesting source of bioactive compounds among which phytosterols should play a major role. The present study is an attempt to investigate the hypocholesterolemic activity of Aloe vera associated with its impact on the reproductive status of juvenile goldfish. Therefore, the short- and long-term effects of feeding supplementary diet containing aloe components (20 mg aloe/g diet; 2 %) on plasma lipids, plasma vitellogenin, and hepatic estrogen receptor α/β1 mRNA levels in goldfish were examined. Results of GC–MS for phytosterols show high abundance of β-sitosterol in freeze-dried powder of Aloe vera whole leaves. Moreover, a 2 % aloe powder dietary supplement was not found estrogenic in juvenile goldfish after either 7- or 30-day treatment, but was consistent in plasma hypocholesterolemic effects following long-term exposure. The present data further support that plasma cholesterol modulation induced by phytosterols may not be related to estrogen-like activity.     

Thiamine requirement of Chinese soft‐shelled turtle,Pelodiscus sinensis (Wiegmann)

The objective of the present study was to estimate the dietary thiamine (vitamin B₁) requirement of juvenile soft‐shelled turtles, Pelodiscus sinensis. Eight experimental diets containing 0, 1.90, 3.63, 5.65, 7.51, 9.62, 11.37 and 13.64 mg thiamine/kg diet were fed to 160 soft‐shelled turtles reared individually for 10 weeks. The turtles had an average weight of 5.33 ± 0.21 g. Among all the dietary groups, weight gain, feed utilization and tissue thiamine were the lowest in the turtles fed with a thiamine‐free diet. The variables increased when dietary thiamine increased and then levelled off beyond 3.63 mg/kg diet. In contrast, plasma pyruvate and lactate concentrations in turtles decreased when dietary thiamine increased and then levelled off beyond 3.63 and 5.65 mg/kg diet respectively. Estimation using a broken‐line regression model, the vitamin B₁ requirement of soft‐shelled turtles was 3.2 mg thiamine/kg diet based on weight gain and plasma lactate concentration. When tissue thiamine and plasma pyruvate concentrations were used as the indicators, the estimated requirement was 5.4–6.5 mg/kg diet.     

Effect of vitamin C dietary supplementation in reducing the alterations induced by fenitrothion in Oreochromis niloticus

The objective of this study was to investigate the dietary effect of vitamin C in amelioration some of studied alterations induced by fenitrothion in Nile tilapia (Oreochromis niloticus). Nile tilapia was exposed to sub-lethal concentration of fenitrothion 0.04 mg/l (96 h LC₅₀ value was 0.8 mg/l), and basal diet was supplemented with two different dose of vitamin C (500 and 1200 mg/kg B wt/day) for 30 days. Vitamin C supplemented groups showed significant decrease in plasma cortisol and glucose level, enzymes activity of liver and gills (catalase, glutathione-S-transferase and superoxide dismutase) and % of tail DNA damage compared to exposed group. Moreover, fish revealed significant increase in total plasma protein, albumin, globulin and A/G ratio. High dose of vitamin C dietary supplementation (1200 mg/kg B wt/day) returns these parameters to its normal levels with no significant difference compared to non exposed control group. These results indicated that incorporation of high dose of vitamin C (1200 mg) in aqua feed for 30 days could be potentially less expensive and effective in reducing the alterations induced by fenitrothion in Nile tilapia.     

仿刺参幼参对饲料中维生素B6需求量的研究

为研究仿刺参幼参对维生素B_6的最适需求量,配制维生素B_6实测含量分别为1.23、5.29、9.35、17.47、33.71和66.17 mg/kg的6组实验饲料D1、D2、D3、D4、D5和D6组,饲喂初始体质量为(12.23±0.11) g的仿刺参幼参12周。结果显示,①随着维生素B_6含量的增加,实验仿刺参的增重率、特定生长率均先升后降,在D5组达到最高值;体壁粗蛋白含量先升后降,D6组显著低于其他组;D1组粗脂肪含量显著低于其他组;②体腔液中葡萄糖-6-磷酸脱氢酶、异柠檬酸脱氢酶、乙酰辅酶A羧化酶及一氧化氮合酶活性均先升后降,D6组显著低于其他组;③随着饲料中维生素B_6含量的增加,肠道蛋白酶及淀粉酶活性显著升高,纤维素酶活性显著降低,肠壁厚度及绒毛长度均显著升高。以增重率为评价指标,经一元二次回归分析得出仿刺参幼参饲料中维生素B_6的适宜需求量为45 mg/kg。     

Antibacterial properties of anthraquinones extracted from rhubarb against Aeromonas hydrophila

Antibacterial properties of crude extract from rhubarb and its major bioactive compounds against Aeromonas hydrophila were assayed. Major bioactive compounds (anthraquinone derivatives) in rhubarb collected from different cultivation areas were determined by ultra-performance liquid chromatography (UPLC); the antibacterial activity [minimum inhibitory concentration (MIC)] of rhubarb was positively related to the anthraquinone content (r = 0.9306, P < 0.01). The MIC values of five anthraquinones against A. hydrophila were found to be in the range 50–200 μg/ml. Action-mode studies showed that anthraquinones (emodin) inhibits cellular functions by binding to cell DNA after penetrating the cell membrane, resulting in cell death. The present study suggests that anthraquinones extracted from rhubarb have potential use as antimicrobials for control of A. hydrophila.     

Involvement of Gonadotropin-Releasing Hormone in Thyroxine Release in Three different forms of Teleost Fish: Barfin Founder, Masu Salmon and Goldfish

Effects of gonadotropin-releasing hormone (GnRH) on thyroxine (T₄) release in vivo and in vitro were studied in barfin flounder Verasper moseri, masu salmon Oncorhynchus masou and goldfish Carassius auratus. Seabream GnRH (sbGnRH) at a dose of 200 ng/50 g body weight (BW) significantly increased plasma T₄ levels 1 h after the in vivo injection in the barfin flounder, but thereafter the levels normalized. Salmon GnRH (sGnRH) significantly increased plasma T₄ levels l h after the injection with a significant return to initial levels in male masu salmon and male goldfish. In contrast, sGnRH and cGnRH-II in barfin flounder, and cGnRH-II in male masu salmon and male goldfish were not effective in stimulating T₄ release. To clarify direct involvement of GnRH in T₄ release, dissected lower jaw including scattered thyroid follicles was incubated with sbGnRH (1 μg/well) in barfin flounder, and with two doses (0.1 and 1 μg/well) of sGnRH in masu salmon and goldfish in vitro. T₄ concentrations of control were stable during 24 h. Incubation of lower jaw with high dose (1 μg/well) of GnRH significantly (P<0.05) increased T₄ concentrations of incubation medium at 1 h in all experimental fishes. These results indicate that direct stimulation of T₄ secretion by GnRH occurs widely in teleost fish.