Selection of lactic acid bacteria as candidate probiotics for Vibrio parahaemolyticus depuration in pacific oysters (Crassostrea gigas)

The application of probiotics in food is now widespread and is widely accepted by consumers. Lactic Acid Bacteria (LAB) were isolated from traditional salted fish and then characterized by its ability to inhibit Vibrio parahaemolyticus growth using minimal inhibitory concentration tests. Five out of these strains were identified as Lactobacillus delbrueckii, Lactobacillus casei and three Lactobacillus gasseri by PCR using 16S and 23S rRNA genes. Antibiofilm activity of Lactobacillus spp. extracts were also tested in 96 polystyrene plates. A potential antibiofilm effect was demonstrated as most LAB. Although most LAB extract were able to eradicate pre‐formed biofilm, results demonstrated that five Lactobacillus spp. exhibited a stronger inhibitory effect against V. parahaemolyticus in infected oysters. Vibrio parahaemolyticus viable cells number declined from 10⁶ UFC to 10⁴ UFC after 3 days of incubation with Lactobacillus spp. Probiotic applications, in biological control of seafood associated pathogens can be an alternative solution, providing consumer with a product of good quality owing to the use of non‐toxic compounds. Based on our results, LAB could be used as a bioprotective culture in oyster's depuration to prevent V. parahaemolyticus growth.     

卤虫无节幼体的活体消毒试验

卤虫无节幼体是培育海水苗种食物链的重要一环,针对卤虫卵孵化中高温、高密度造成的无节幼体细菌微生物含量过高从而影响育苗成活率这一问题,进行了杀菌试验,结果表明:随着杀菌时间延长到60 min,使用二氧化氯和复合硖杀菌的卤虫无节幼体活力下降,使用紫外线的卤虫无节幼体活力正常;紫外线照射对卤虫无节幼体暂养水的细菌菌落杀灭效果...     

Evaluations of lactic acid bacteria as probiotics for juvenile seabass Lates calcarifer

Lactic acid bacteria (LAB) were isolated from adult, wild‐caught and farmed seabass (Lates calcarifer) intestines for evaluation as possible probiotics using the well agar diffusion method. Five LAB isolates (designated as LAB‐1–5) were found to inhibit Aeromonas hydrophila, a known seabass pathogen. Median lethal concentrations (LC₅₀) of A. hydrophila on juvenile seabass were measured in aquaria. Median lethal concentration values of 7.76, 7.47 and 7.26 log₁₀ CFU mL^?1 for 72, 96 and 120 h, respectively, were found. Juvenile seabass (0.6±0.2 g) were cultured in aquaria and fed individual LAB‐1–5 fortified feeds with 7 log₁₀ CFU g^?1 LAB. Seabass fed LAB‐4 fortified feed had significantly greater growth (P<0.05) than fish fed other feeds. Seabass fed LAB‐4 also had greater survival, but this was non‐significant (P<0.05). Challenge tests of LAB‐4 fed seabass with A. hydrophila at ～7 log₁₀CFU mL^?1 yielded significantly greater survival compared with control seabass (P<0.05). Aeromonas hydrophila infections in seabass were confirmed by observing disease manifestation and by immunohistochemistry techniques. LAB‐4 was preliminarily identified using lactic acid analysis, biochemical and physical characteristics. It was further identified using 16S rDNA sequencing. LAB‐4 was identified as Weissella confusa (identity of 99%). GenBank accession number for the 16S rDNA sequence for LAB‐4 was AB023241.     

不同强化物质对卤虫体内游离氨基酸含量的影响

以卤虫无节幼体作为试验材料,研究不同强化物质对卤虫体内游离氨基酸(FAA)含量的影响。试验分为4组,分别强化赖氨酸(Lys)、裂壶藻Schizochytrium、赖氨酸加裂壶藻以及空白对照组,试验进行16h,每4h取样1次。结果显示,试验结束时,赖氨酸组的卤虫体内游离Lys含量显著高于其他3个组,且较强化前相比增加了4倍多,裂壶藻组的含量最低。除苏氨酸(Thr)外,赖氨酸组其他游离必需氨基酸以及天冬氨酸(Asp)、丝氨酸(Ser)、甘氨酸(Gly)、酪氨酸(Tyr)含量都显著高于其他3个组。赖氨酸加裂壶藻组,Thr、Ser、Gly、丙氨酸(Ala)、组氨酸(His)和脯氨酸(Pro)含量均有不同程度的减少,其余组的卤虫,只有Thr含量减少,其他几种FAA水平均增加。试验过程中FAA水平随时间的变化趋势,空白组和赖氨酸加裂壶藻组相同,除Thr外,其余FAA含量都是在4h时增高,8h时下降,12h时升高;而在赖氨酸组和裂壶藻组的卤虫体内,这些FAA含量是前8h升高,12h时降低,16h时又升高。     

Uptake and metabolism of a particulate form of ascorbic acid by Artemia nauplii and juveniles

A study was conducted to establish whether a particulate form of ascorbic acid (AA), ascorbyl‐2‐phosphate (A2P), could be used to enrich Artemia. In the first experiment, we examined the efficiency of A2P conversion to and maintenance of AA by juvenile Artemia (1.5 mm, 5‐day‐old) held at 9000 L^?1 and 28 °C for 24 h. Maximal uptake and assimilation was >10 000 μg AA g^?1 dry weight (dw) (representing >1%Artemia dw) at enrichment rates of ≥1.2 g A2P L^?1. In the second experiment, a similar biomass of instar II/III nauplii (1 mm, 2‐day‐old) and juvenile (2.5 mm, 8‐day‐old) Artemia were enriched for 6 or 24 h at 28 °C before starvation for 6 or 24 h at 18 or 28 °C. At 0 h and after 6 and 24 h enrichment, AA levels were 485, 3468 and 11 080 μg g^?1 dw in nauplii and 122, 4286 and 12 470 μg g^?1 dw in juveniles. When Artemia nauplii or juveniles were enriched for 6 h and starved for 6 h at 18 or 28 °C, there was no significant reduction in AA. Continuation of starvation to 24 h at 18 and 28 °C reduced the level of AA to 3367 and 2482 μg g^?1 dw in nauplii and 3068 and 2286 μg g^?1 dw in juveniles. After 24 h enrichment, 6 h of starvation at 18 and 28 °C reduced AA to 8847 and 7899 μg g^?1 dw in nauplii and to 9053 and 8199 μg g^?1 dw in juveniles. Continuation of starvation to 24 h at 18 and 28 °C further reduced AA levels in nauplii to 6977 and 4078 μg g^?1 dw and to 7583 and 5114 μg g^?1 dw in juveniles. This study demonstrated that A2P could be assimilated as AA in the body tissue of different‐sized Artemia in a dose‐dependant manner and AA was depleted during starvation depending on time and temperature.     

Bioencapsulation and pharmacokinetic study of oxolinic acid in rotifers (Brachionus plicatilis), Artemia franciscana nauplii and metanauplii

The uptake of oxolinic acid by the rotifer Brachionus plicatilis, Artemia franciscana nauplii and metanauplii was studied as a function of its concentration in the enrichment medium and the duration of the enrichment period. An emulsion containing 5, 10, 20 or 30% (w/w) oxolinic acid was administered and the enrichment period lasted 4, 8, 12 or 36 h. Highest incorporation of oxolinic acid was achieved using a 20% emulsion and a 12 h enrichment for rotifers (205.05 ± 17.1 μg g^?1 dry weight), a 24 h enrichment for nauplii (2528.8 ± 254.6 μg g^?1 dry weight), and an 8 h enrichment for metanauplii (1236.58 ± 22.9 μg g^?1 dry weight). Higher concentrations of oxolinic acid in the enrichment emulsion or longer enrichment times resulted in decreased survival. Two hours post enrichment the contents of the drug appeared significantly decreased. The concentration data of oxolinic acid were best fit to a two phase exponential elimination model, the first phase elimination half‐life (t_1/2α) being 1.86, 1.08 and 1.74 and the terminal phase elimination half‐life (t_1/2β) 26.83, 29.67 and 17.48 in rotifers, nauplii and metanauplii correspondingly. Enrichment with an emulsion containing 20% oxolinic acid is recommended employing a duration of 12, 24, or 8 h enrichment for rotifers, nauplii and metanauplii respectively, while enriched carriers should be used shortly after enrichment.     

光合细菌在卤虫饵料中的应用

以螺旋藻作为卤虫的饵料,在卤虫培养液中添加不同体积分数的光合细菌(0,0 1%,0 3%,0 5%),经15d的养殖,卤虫的成活率、内禀生长率、粗蛋白含量均比对照组显著增加。卤虫必需氨基酸的总量和氨基酸总量添加组与对照组差异不大,成体卤虫均可作为河蟹蟹苗的优质蛋白源。     

不同饵料对中华绒螯蟹幼体发育和存活的影响

采用高度不饱和脂肪酸（ＨＵＦＡ）营养强化的轮虫、轮虫与卤虫组织投喂中华绒螯蟹幼体,以各项幼体的存活率为评判指标,对不同饵料及其组合的投喂效果进行了探讨。研究结果表明：轮虫是中华绒螯解早期幼体（Ⅰ、Ⅱ期）的适口饵料,幼体存活率随轮虫密度的增加而逐渐上升,但当轮虫数量超过最适密度时,幼体存活率反而有所下降。其中,未强化的轮虫达投喂密度为６０ｉｎｄ．ｍＬ＾－１,强化轮虫最适密度为４０ｉｎｄ．ｍＬ＾－１。Ｚａｏ状Ⅰ、Ⅱ期投喂４０ｉｎｄ．ｍＬ＾－１轮虫,从Ⅲ期开始投喂１０ｉｎｇ．ｍＬ＾－１卤虫无节幼体,能较好地满足中华绒螯蟹幼体发育的营养需求,提高大眼幼体的存活率。表明强化幼体饵料ＨＵＦＡ特别是ＥＰＡ和ＤＨＡ能有效地促进幼体的发育与存活和脱壳率。     

Feeding of scleractinian coral, Galaxea fascicularis, on Artemia salina nauplii in captivity

Satisfying nutrient requirement of corals is still a major constraint for maintaining corals in marine aquariums. Corals are polytrophic in nature. Heterotrophic feeding on zooplankton is one of the corals’ strategies to overcome nutrient deficiency. Artemia salina nauplii are commonly used as biocarriers for many fish larvae in aquaculture and can also serve as a biocarrier for coral in aquariums, provided coral acceptability, optimal feeding rate, and digestibility of the nauplii are well understood. Feeding rate and digestibility of coral fed on A. salina nauplii at 100, 2,000, 4,000, 6,000, and 10,000 ind. l⁻¹ under light and dark conditions was assessed in this study. The maximum feeding rates of Galaxea fascicularis under light and dark conditions was 113.6 ind. polyp⁻¹ h⁻¹ and 76.9 ind. polyp⁻¹ h⁻¹, respectively. The daily feeding rates of G. fascicularis varies and depends on nauplii density. Light plays an important role in coral feeding. Nevertheless, the quantity of A. salina nauplii consumed by the coral under light and dark conditions was not significantly different (P > 0.05). A. salina nauplii are well accepted by G. fascicularis. Complete nauplii digestion was observed after 180 min. Digestibility of A. salina nauplii by G. fascicularis was positively correlated with digestion time.     

Encapsulation capacity of Artemia nauplii with customized probiotics for use in the cultivation of western king prawns (Penaeus latisulcatus Kishinouye, 1896)

The encapsulation capacity of Artemia nauplii with customized probiotics Pseudomonas synxantha and Pseudomonas aeruginosa for use in the cultivation of western king prawns (Penaeus latisulcatus) was investigated. Seven trials were conducted to investigate this encapsulation capacity in terms of Artemia survival and probiotic load in Artemia. Newly hatched Artemia nauplii at 250 nauplii mL^?1 were fed individual probiotics at 0, 10³, 10⁵ and 10⁷ colony‐forming units (CFU) per millilitre, and mixtures of these two probiotics (10⁵ CFU mL^?1) at 30:70, 50:50 or 70:30 v/v in a medium of ozonated water (OW), tryptone soya broth (TSB), and a mixture of these media. The appropriate medium for encapsulation of probiotics by Artemia nauplii was the mixture of OW and TSB at 75:25 v/v; whereas, the use of OW or TSB alone was not effective. Artemia nauplii most effectively encapsulated the customized probiotics at 10⁵ CFU mL^?1. The results indicates that the encapsulation of Artemia nauplii is optimized by using a combination of P. synxantha and P. aeruginosa at 50:50 v/v in a media mixture of OW and TSB at 75: 25 v/v. Artemia should be harvested at 48 h when survival is still high (78%) and the probiotic load in Artemia is high (3 × 10⁴ CFU nauplius^?1).     

饵料卤虫的营养强化对黑斑口虾蛄(Oratosquilla kempi) 幼体消化酶活力及其抗逆性的影响

用扁藻、酵母和"鱼油 酵母"强化3种方式处理的卤虫无节幼体投喂黑斑口虾蛄幼体,对各组黑斑口虾蛄幼体消化酶活力和抗逆性差别分析。结果显示,经过"鱼油 酵母"强化的幼体对氨氮、福尔马林、饥饿和盐度的耐受性均强于对照组。经过扁藻和"鱼油 酵母"强化的第Ⅲ相幼体脂肪酶活力是对照组的3倍,经过酵母和"鱼油 酵母"强化的仔虾蛄幼体脂肪酶活力较高。在第Ⅲ相幼体中淀粉酶活力为扁藻>酵母>对照>"鱼油 酵母"强化,仔虾蛄中酶活力为扁藻>对照>酵母>"鱼油 酵母"强化。在酵母强化第Ⅲ相幼体和对照组仔虾蛄中纤维素酶活力最高。第Ⅲ相幼体对照组的胃蛋白酶和类胰蛋白酶活力均为最高,而"鱼油 酵母"强化仔虾蛄幼体的胃蛋白酶和类胰蛋白酶活力均高于对照组。黑斑口虾蛄幼体消化酶活性和抗逆性能力的变化趋势与其成活率、变态率、生长速度、EPA和DHA含量变化趋势相同,可以作为评价饵料质量好坏的重要指标。     

International study on Artemia XXIV. Cold storage of live Artemia nauplii from various geographical sources: Potentials and limits in aquaculture

Freshly-hatched Artemia nauplii from various geographical sources survived storage in a refrigerator (2–4°C) at densities of 2000 per ml and above. Except for Artemia from Chaplin Lake and Buenos Aires, naupliar viability was very high even after 48 h storage, and did not decrease significantly after a 24 h post-storage transfer to 25°C. Neither the naupliar dry weight nor biochemical composition changed significantly during refrigeration for most strains tested. Comparative culture-tests with stored and freshly-hatched nauplii as food for juvenile marine mysids Mysidopsis bahia M. and larval carp Cyprinus carpio L. revealed similar production performances.     

牛磺酸对卤虫无节幼体体成分的营养强化作用

为探讨牛磺酸强化卤虫无节幼体对其体成分的影响,试验分别以0、10、100、1 000mg/L浓度的牛磺酸强化卤虫无节幼体12h和24h后,采用国标法检测卤虫无节幼体水分、粗蛋白、粗脂肪、粗灰分、钙和磷的含量。试验结果显示:100mg/L牛磺酸强化卤虫无节幼体12h能显著提高其粗蛋白(P0.05)、粗脂肪、钙和磷的含量,显著降低其粗灰分含量(P0.05);10mg/L牛磺酸强化卤虫无节幼体24h能显著提高其粗蛋白和钙的含量(P0.05),显著降低其粗脂肪和粗灰分含量(P0.05);100mg/L牛磺酸强化卤虫无节幼体24h能显著提高其粗蛋白、钙和磷的含量(P0.05),显著减低其粗脂肪和粗灰分含量(P0.05)。表明:本试验条件下10mg/L牛磺酸强化卤虫无节幼体24h能显著改善卤虫无节幼体的体成分。     

珊瑚对型轮虫和丰年虫无节幼体的捕食机制初探

为探讨珊瑚对生物活饵料的捕食机制,选取美丽鹿角珊瑚(Acropora formosa)和佳丽鹿角珊瑚(Acropora pulchra)为实验对象,以轮虫(SS-type Rotifer)和丰年虫(Artemia salina)无节幼体为生物活饵料进行投喂。通过对投喂饵料量、残余量,以及差值进行统计,从而探讨珊瑚的摄食情况及摄食偏好。结果表明:超小型轮虫平均剩余量8.16×104个,P=0.012(P0.05),实验前后轮虫密度有显著差异。丰年虫无节幼体平均剩余量2.3×103个,P=0.01(P0.05),实验前后丰年虫密度有显著性差异。超小型轮虫和丰年虫无节幼体可以是珊瑚的生物活饵料。     

抗嗜水气单胞菌NJ-1感染的乳酸菌筛选及其免疫调节作用

为了筛选能提高水产动物对嗜水气单胞菌抗病性的益生菌,并研究其免疫调节的作用机制。将5种乳酸菌与斑马鱼ZF-4细胞共培养后通过荧光定量PCR方法检测细胞因子NFκB、TNF-α和IL-10的表达量,并用流式细胞仪检测被嗜水气单胞菌NJ-1攻毒后的细胞凋亡率来筛选对ZF-4细胞保护性最显著的乳酸菌,将其添加至模式生物斑马鱼饲料中饲喂28 d后,用嗜水气单胞菌NJ-1攻毒并检测斑马鱼肠道中促炎性细胞因子TNF-α、IL-1β和抗炎性细胞因子IL-10的表达情况,并计算斑马鱼被攻毒后的累积死亡率。结果显示,5种乳酸菌分别与细胞共培养后,芽孢乳酸菌09.712能明显提高ZF-4细胞NFκB、TNF-α和IL-10的表达量,攻毒后细胞凋亡率降低至23.36%,与其他组相比存在显著性差异,所以选取芽孢乳酸菌09.712用于下一步实验。体内实验显示,攻毒后第1天,与对照组相比,芽孢乳酸菌09.712能显著促进斑马鱼肠道内IL-1β、TNF-α和IL-10的表达量,且随时间变化表达量减少;其中攻毒后第3天,菌饲喂组IL-1β表达量比对照组低,而IL-10表达量明显比对照组高。此外,芽孢乳酸菌09.712的饲喂显著降低斑马鱼攻毒后的死亡率。研究表明,芽孢乳酸菌09.712在体外能显著提高对细胞的保护性,在体内能诱导斑马鱼产生先天免疫应答,提高斑马鱼的免疫力,可作为预防水产动物疾病的理想选择,也为阐明乳酸菌先天性免疫调节作用提供理论基础。     

Enrichment of Artemia nauplii in PUFA, phospholipids, and water-soluble nutrients using liposomes

Different liposome formulations, includingseveral combinations of membrane composition,type of vesicle (multilamellar and largeunilamellar vesicles), preparation method, andvehiculated nutrient, have been assayed asbioencapsulation products to enrich Artemia nauplii with nutrients for feeding fish larvae.The stability of the liposome preparationsunder conditions of use as enrichment producthas been tested using water soluble fluorescentmarkers as leakage indicators. The content ofthe fatty acids and lipid classesbioencapsulated in Artemia nauplii withliposomes has been analyzed by gas and thinlayer chromatography, respectively, andcompared with other enrichment products. Theeffect of the liposome enriched Artemianauplii used as food for fish larvae has beenevaluated in sea bass cultures. Liposomes withhigh content in polyunsaturated fatty acidsleak out more than 50% of their aqueous phasein less than 2 hours, unless they arestabilized with cholesterol and formed as largeunilamellar vesicles. Such vesicles hold70% of the encapsulated material for 8 hours.Liposome enriched nauplii in this study reflectthe influence of the enrichment products,however, they are far from the commercialemulsion (Super Selco) in terms ofdocosahexaenoic acid content, except for thenauplii enriched with liposomes made of purekrill phospholipid extract by the method ofdetergent solubilization. The liposome enrichednauplii show a higher amount of polar lipids incontrast to the feed enriched with emulsions.The larvae fed liposome enriched nauplii haveonly a slightly lower docosahexaenoic acidcontent than those fed emulsion enrichednauplii. The results obtained confirm thesuitable potential use of liposomes as foodsupplement in larviculture. Problems andadvantages are discussed.     

乳酸菌在青贮中的应用及研究进展

青贮饲料是一个复杂的微生物共生体系,主要包括乳酸菌、酵母菌、芽孢杆菌、乙酸菌、梭状芽饱杆菌等,其中以乳酸菌的数量和种类最多,也是在青贮饲料发酵中起主要作用的微生物。着重介绍了乳酸菌微生物青贮剂的种类、选作微生物青贮剂的乳酸菌需符合的条件,阐述了乳酸菌微生物青贮剂的作用机制、添加效果以及影响因素,并对乳酸菌微生物青贮剂近年的研究进展和应用现状进行了总结,指出了乳酸菌微生物青贮剂的未来发展趋势将由单一型向复合型方向发展,同时需将现代基因工程、遗传工程技术引入乳酸菌微生物青贮剂,以开发新型制剂。     

Preliminary assessment of in vivo safety of potentially probiotic lactic acid bacteria for American bullfrog culture

The effect of the administration of beneficial lactic acid bacteria (LAB): Lactococcus lactis CRL 1584, L. lactis CRL 1827, Enterococcus gallinarum CRL 1826 and combined CRL 1584+ CRL 1826 on the development of bullfrog embryos from the hatching stage until 31 days (tadpole) was evaluated. In vitro assays indicated that 10³, 10⁶ and 10⁹ CFU mL^?1 single LAB strains remained viable until 24 h in 10% Ringer. Around 10⁹ CFU mL^?1 LAB (individually and combined) were used in an experimental design built to evaluate their effect when administrated at different intervals (three 7‐day cycles with 5‐day rest periods in between) to embryos until day 31. The highest potentially beneficial population (LAB) numbers were detected in the LAB‐treated groups. All the LAB‐treatments increased it and were significantly higher than the controls. Although the highest, potentially pathogenic, population (Red‐Leg Syndrome‐RLS‐related pathogens) numbers were detected in the control and the lowest in the CRL 1584+ CRL 1826‐treated group, they did not differ significantly. Stereoscopic studies showed no malformations in any LAB‐treated group and all the specimens reached the same stage of their biological cycle with a survival >94%. The histological structure of target organs for RLS‐associated pathogens (intestine and skin) and stomach was not affected and the spleen was developed. Only the LAB‐treated groups showed microorganisms associated with the intestinal mucus, without inflammatory reaction in the lamina propria. This is the first report on the evaluation of the absence of adverse effects after LAB administration to bullfrog embryos using indigenous microorganisms.