Selection of lactic acid bacteria as candidate probiotics and in vivo test on Artemia nauplii

Lactic acid bacteria (LAB) were isolated from rotifer cultures in a marine hatchery to search for potential probiotics for marine animals. Fifteen strains were first selected among a total of 55, according to antibacterial activity against Vibrio sp. Among eight strains identified as Lactobacillus casei, four were highly adhesive, suggesting some ability for surface colonization. The other strains were identified as Lactobacillus plantarum, Lactobacillus dextrinicus, and Leuconostoc sp. To validate probiotic potential, Artemia were challenged against pathogenic Vibrio alginolyticus, with or without one of six selected LAB strains. The six strains protected Artemia against the pathogen, to some extent on condition that nutrient enrichment was provided. La. casei BR51 and X2 were preferred, as they were efficient even in the absence of nutrient supply. La. casei X2 was finally selected as candidate probiotic, due to the best growth performances of Artemia, with or without the pathogen.     

Biochemical and Sensory Characteristics of Whole Carp Inoculated With Autochthonous Starter Cultures

In this study, Lactobacillus plantarum 120, Lactobacillus plantarum 145, and Pediococcus pentosaceus 220, which exhibit optimal properties among those isolated from the traditional fermented fish products, were selected as the starter cultures for the preparation of fermented whole carp. During the fermentation, samples inoculated with different single starter cultures showed rapid growth of lactic acid bacteria (LAB), decline in pH, suppression in the growth of main microflora, as well as increase of thiobarbituric acid reactive substances (TBARS) values and total volatile base nitrogen (TVB-N). Quantities of nonprotein nitrogen (NPN) and free amino acids (FAA) were higher in whole carp with starter cultures compared to those in the control. Severe hydrolysis of protein was observed utilizing sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis during fermentation. Moreover, sensory evaluation indicated the higher acceptability of fermented products. The results show that inoculation with the autochthonous starter cultures reduced fermentation time and improved the quality of samples. Therefore, isolates of the strains could be used as potential starters for the fermentation of whole carp.     

Effect of potential probiotic bacteria on growth and survival of tilapia Oreochromis niloticus L., cultured in the laboratory under high density and suboptimum temperature

This study examined the effect of potential probiotic bacteria on growth and survival of the tilapia Oreochromis niloticus , under high density and suboptimum temperature. Presumptive Bacillus and lactic acid bacteria (LAB) were isolated from tilapia and from their culture system and were characterized for haemolytic and enzymatic activity, and antagonism against Vibrio . Selected strains were included in the diet of juvenile tilapia and evaluated during a 134-day assay. The experiment was conducted with four treatments: (1) fish fed with commercial feed plus Dry Oil^®; (2) fish fed with commercial feed plus LAB; (3) fish with bacilli in water; (4) fish with a mixture of treatments 2 and 3. Tilapias in all treatments, including bacteria, grew significantly better than fish fed with commercial feed plus Dry Oil^® (control group). Survival was similar in all treatments. The physicochemical parameters of the culture system were maintained within the optimal ranges for the species, with the exception of temperature (19.9–24.82 °C). Animals fed diet supplemented with bacilli and LAB had good survival and the best growth performance, suggesting that bacteria are appropriate growth-stimulating additives in tilapia cultivation.     

乳酸菌对牙鲆稚鱼养殖水体和肠道菌群的影响

在牙鲆稚鱼中投喂添加由单一鼠李糖乳杆菌P15制备的微生态制剂和黄霉素,在60d的投喂期内,采用平板计数法检测好氧性异养菌的总数、总弧菌数和乳酸菌的数量。结果表明,在投喂菌液和冻干菌粉后,养殖水体和牙鲆肠道的乳酸菌数均呈上升趋势,在30d后乳酸菌数量达到稳定并在肠道内定植。同时,由于乳酸菌的抑制作用,弧菌的数量下降,以肠道中的弧菌最明显。乳酸菌对养殖水体和牙鲆肠道的好氧性异养菌没任何影响。乳酸菌对养殖水体和肠道菌群的影响与抗生素具有相近的效果,表明乳酸菌作为饲料添加剂可以取代抗生素应用在牙鲆的养殖中。     

Enhancement of rotifer (Brachionus plicatilis) growth by using terrestrial lactic acid bacteria

One of the pathways for the entry of bacteria, both pathogenic and probiotic, into the larvae of fish hatcheries is via live prey. As a preventive measure against infections, live prey may be cultured, supplementing the food with probiotics. Some lactic acid bacteria (LAB) have been successfully used in the larviculture. In this study, the nutritional effect of seven terrestrial LAB has been studied regarding the growth of the rotifer Brachionus plicatilis. The cultures were carried out without partial renewal of the culture medium, feeding the rotifers on baker's yeast and adding some of the species of bacteria. In all cases, the addition of the bacteria increased both the specific maximum growth rate and the maximum density obtainable in the cultures. However, the best results were obtained with the addition of Lactococcus casei ssp. casei, Pediococcus acidilactici, or Lactobacillus lactis spp lactis. The rates of growth obtained with the individual or joint addition of these three bacteria were 8–13 times greater than those obtained with the control cultures after 4–5 days' culture. In this study, a series of kinetic models has been applied (logistic modified—Gompertz, logistic–logistic, and generalised logistic) which describes the experimental data, obtaining a set of parameters of biological significance which facilitate the optimisation of the use of these bacterial strains in the mass production of rotifers.     

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.     

Effects of Lactobacillus pentosus on the growth performance,digestive enzyme and disease resistance of white shrimp,Litopenaeus vannamei (Boone, 1931)

The objective of this study was to evaluate the probiotic properties of lactic acid bacteria (LAB) strains isolated from digestive tract of white shrimp Litopenaeus vannamei. Eighteen LAB colonies were isolated and one bacterium was found capable of producing three extracellular enzymes (protease, cellulose and lipase) simultaneously and exhibited antagonistic activity against shrimp pathogens (Vibrio vulnificus, V. rotiferianus and V. campbellii). The putative probiotic strain AS13 was identified as Lactobacillus pentosus based on 16S rRNA sequencing. The L. vannamei were fed diet containing 0 (control), 10⁶, 10⁷ and 10⁸ CFU g^?1 bacterial cells of AS13 for 28 days. The results showed that supplementation of L. pentosus significantly improved the growth performance and feed utilization in the treated groups over the control. Similarly, digestive enzyme activities were elevated in the intestines of treated groups. Moreover, feeding of supplemented diets containing AS13 significantly reduced the mortality rate caused by pathogenic Vibrio species (V. vulnificus, V. rotiferianus and V. campbellii). Our results indicated L. pentosus AS13 addition at 10⁷ CFU g^?1 can effectively enhance the growth performance, feed utilization, digestive enzymes and disease resistance of L. vannamei in the laboratory condition.     

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.     

Effects of dietary mono‐ and multiprobiotic strains on growth performance,gut bacteria and body composition of Javanese carp (Puntius gonionotus,Bleeker 1850)

This study investigated the effects of dietary mono‐ and multiprobiotic strains of Enterococcus faecalis, Lactobacillus fermentum and Leuconostoc mesenteroides on growth performance, intestinal bacteria and body composition of Javanese carp (Puntius gonionotus). Moreover, the effect of storage temperature on lactic acid bacteria (LAB) viability in the feed was studied. Compared to the control group, significant (P < 0.05) effect on weight gain, feed conversion ratio, specific growth rate and protein efficiency ratio of probiotic fed Javanese carp was observed. Inclusion of individual LAB strains in the diet resulted in significantly improved growth performances of Javanese carps compared to fish fed a diet supplemented LAB mixture. The inclusion level of E. faecalis at 10⁷ colony‐forming unit (cfu) g⁻¹ in the diet revealed higher growth performance compared to other treatments. The population levels of LAB in the fish intestine were significantly increased while the levels of Gram‐negative bacteria were significantly decreased compared to the control. No significant variations were observed in the chemical composition of the carcass by probiotics. The viability of LAB in feed was higher when stored at 4 °C compared to 25 °C. It is suggested that E. faecalis has a greater potential for use in improving Javanese carp culture outcomes.     

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。     

In vitro and in vivo antibacterial activity of shrimp chitosan against some intestinal bacteria

ABSTRACT:   The effects of shrimp chitosan with deacetylation degrees (DD) of 50%, 70% and 95% (DD50, DD70, DD95) on the growth of the intestinal bacteria were investigated in vitro in the laboratory media, and in vivo by an oral feeding test using hamsters as the animal model. The antibacterial activities of these chitosan products against one strain of pathogenic Clostridium perfringens and 13 strains of probiotics, including seven strains of Lactobacillus , and six strains of Bifidobacterium were evaluated. In vitro , the antibacterial activities of DD95 and DD70 were much higher than that of DD50. The strains of probiotics were more resistant to chitosan than the pathogen of C .  perfringens . The minimal lethal concentration for DD95 against C. perfringens was 250 p.p.m., whereas the survival percentages for most probiotics tested were above 90% for DD95 at 500 p.p.m. The animals were fed on either a control diet, or diets containing powdered chitosan instead of 5% cellulose in the control diet for 4 weeks. The cecal bacterial counts of total aerobes, total anaerobes, lactobacilli, bifidobacteria and clostridia were similar for the control and experimental groups. The reasons for the differences in the antibacterial activity in vitro and in vivo are discussed.     

Effects of synbiotic containing Lactobacillus plantarum 7–40 and galactooligosaccharide on the growth performance of white shrimp,Litopenaeus vannamei

This study aimed to develop a synbiotic combination with probiotic, Lactobacillus plantarum 7–40 and one of three prebiotics, fructooligosaccharide (FOS), galactooligosaccharide (GOS) and mannan oligosaccharide (MOS). The best in vitro growth was observed when probiotic was cultured in the medium containing either FOS or GOS as the sole of carbon source. The analysis of enzyme activity revealed that GOS induced the highest activities of protease and β‐galactosidase of probiotic. Based on the findings, probiotic + GOS were selected as synbiotic to evaluate if it could promote the growth of white shrimp, Litopenaeus vannamei. For this, four diets, including a basal diet with no GOS or probiotic (control), 0.4% GOS (PRE), 10⁸ CFU probiotic kg^?1 (PRO) and 0.4% GOS in combination with 10⁸ CFU probiotic kg^?1 (SYN), were fed to shrimp for 60 days, and then the growth performance, intestinal microbiota (including total Vibrio counts, VBCs; and lactic acid bacteria, LAB) and digestive enzyme (including protease, leu‐aminopeptidase and β‐galactosidase) were evaluated. The weight gain (WG) of shrimp fed the PRO did not significantly differ from those of control (p > .05). Shrimp fed the SYN had significantly higher WG compared with the other treatments (p < .05). In addition, the SYN‐fed shrimp had significantly higher LAB and protease, leu‐aminopeptidase and β‐galactosidase activity (p < .05). The lowest presumptive Vibrio count (VBC) was also observed in intestines of SYN‐fed shrimp. Therefore, we suggested that Lac. plantarum 7–40+ GOS can be used as a synergistic synbiotic for shrimp culture.     

Recovery of Chitin from Antarctic Krill (Euphausia superba) Shell Waste by Microbial Deproteinization and Demineralization

A two-stage fermentation process of deproteinization and demineralization was used to purify chitin from Antarctic krill shell waste. Two bacterial cultures were isolated and tested for the degradation of krill shell waste. According to the morphological examination, physiological tests, and applied molecular techniques conducted, isolates were identified as Bacillus subtilis and Lactobacillus plantarum. At a temperature of 37°C, both strains were cultivated separately in flasks containing krill shell waste. At determined periods of time, deproteinization and demineralization of residuals were measured. For deproteinization tests, the liquid phase fermentation of krill shell waste showed protein removal of 88.9% after 60 h. For demineralization tests, the liquid phase fermentation of krill shell waste showed calcium carbonate removal of 84.6% after 72 h. Fourier transform infrared spectroscopy (FTIR) analysis of chitin prepared by the process was carried out and compared with spectra of samples treated by strong acids and bases.     

Identification of gut‐associated amylase,cellulase and protease‐producing bacteria in three species of Indian major carps

Isolation and enumeration of amylase, cellulase and protease‐producing autochthonous bacteria in the proximal intestine (PI) and distal intestine (DI) of three species of Indian major carps, catla (Catla catla), mrigal (Cirrhinus mrigala) and rohu (Labeo rohita), were investigated using the conventional culture‐based technique. Population levels of amylolytic strains were the highest in the PI of catla and the lowest in the DI of rohu. The highest viable count of cellulase and protease‐producing bacteria was recorded in the DI and PI of mrigal respectively. Among the bacteria isolated, 10 strains (five from PI and five from DI) were selected as potent enzyme producers according to a quantitative enzyme assay. The chosen strains were further identified by 16S rRNA gene sequence analysis. The five strains isolated from catla showed high similarity to Citrobacter sp. clone W2, Enterobacter sp. JA24, Bacillus coagulans strain TR, uncultured bacterial clone Hel3bc04 and Bacillus cereus strain UST2006‐BC004. The four strains isolated from mrigal were most closely related to Bacillus sp. KCd2, uncultured bacterial clone Hel3bd09, B. cereus strain BU040901‐020 and Citrobacter freundii strain YRL11, while the strain isolated from rohu probably belonged to Bacillus sp. GV.     

乳酸菌利用罗非鱼下脚料水解液发酵L-乳酸的探讨

以罗非鱼（Oreochromis spp.）下脚料为原料,采用高温热水抽提法提取其蛋白质作为乳酸菌发酵L-乳酸的氮源。以酵母膏作为对照,探讨了保加利亚乳杆菌（Lactobacillus bulgaricus）、嗜热链球菌（Streptococcus thermophi-lus）和鼠李糖乳杆菌（L.rhamnosus）在罗非鱼下脚料水解液培养基中的生长情况及L-乳酸的产量。结果显示,3种乳酸菌在2种培养基中均能良好地生长,pH下降明显;L-乳酸产量随乳酸菌菌种不同而异,其中鼠李糖乳杆菌在罗非鱼下脚料水解液培养基中发酵10 h后,L-乳酸净产量为3.38 g.L-1,与以酵母膏作为氮源的产酸水平相当。可见罗非鱼下脚料水解液可以替代酵母膏作为L-乳酸发酵的一种低成本氮源。     

唾液乳杆菌NF19的液体发酵条件研究

从舟山海域海水中筛选到一株海洋细菌NF19,经16S rDNA分子鉴定和API鉴定结果显示菌株NF19为唾液乳杆菌属(L.salivarius)的一个种,将序列上传至NCBI获得GenBank登录号为FJ405036。通过对菌株NF19进行发酵条件优化及正交试验,得到菌株NF19的最佳发酵条件为:初始pH7.2,接种量为8%,在120 r/min下20℃培养48 h。此条件下发酵液有最佳抑菌效果。     

Beneficial properties of lactic acid bacteria isolated from a Rana catesbeiana hatchery

This work addresses the selection of potentially probiotic lactic acid bacteria (LAB) to be used in raniculture. Thus, strains belonging to the genera Pediococcus pentosaceus, Leuconostoc mesenteroides, Lactococcus lactis and Enterococcus faecium isolated from a Rana catesbeiana hatchery were evaluated for their inhibitory properties against RLS-associated pathogens ( Proteus vulgaris, Pseudomonas aeruginosa, Staphylococcus epidermidis ) and food-borne bacteria. Cell-free supernatants of LAB strains inhibited the growth of at least one of the pathogens by organic acids, but L. lactis CRL 1584 also produced a bacteriocin-like metabolite. The ability of LAB strains to produce H₂O₂ in MRS+TMB medium was also studied. Seventy-eight to ninety six per cent of the strains showed some level of H₂O₂ production. Moreover, different organic solvents were used to determine the hydrophobicity and Lewis acid/base characteristic of LAB strain surfaces. Most of the strains presented hydrophilic properties, but no acidic or basic surface characters. However, some strains isolated from the skin showed a high degree of hydrophobicity and basic components in the cell surface due to their adhesion to chloroform. These properties were not observed in LAB from balanced feed and freshwater. Taking into account general guidelines and the beneficial properties studied, five strains were selected as potential candidates to be included in a probiotic for raniculture.     

Comparison of Lactic Acid Bacteria Fermentation with Acid Treatments for Chitosan Production from Shrimp Waste

The traditional procedure for chitosan production involves use of a strong acid (HCl) for demineralization of chitin. This study reports application of a mixed culture of lactic acid bacteria (Lactobacillus plantarum, Lactobacillus acidophilus, and Lactobacillus lactis) fermentation in demineralization of chitin for chitosan production from shrimp waste. Chitosan produced from shrimp waste with lactic acid bacteria fermentation at 30°C for 72 h or 1 M lactic acid treatment at room temperature for 2 h followed by alkaline treatments (0.5 M NaOH at 25°C for 4 h followed by 12.5 M NaOH at 70°C for 10 h) contained very low protein (0.8–1.1%) and ash (<0.01%) contents with the same solubility (100%) as those of chitosan produced with hydrochloric acid treatment. Lactic acid bacteria fermentation and lactic acid treatment may be utilized in chitosan production from shrimp waste to reduce use of strong chemicals.     

Extracellular protease production of bacteriolytic bacteria isolated from marine environments

ABSTRACT:   Fourteen bacterial strains isolated from marine environments exhibited antagonistic action against a wide range of bacteria including Vibrio spp. A double layer agar method was used for preliminary screening to determine the relative degree of growth inhibition or bacteriolysis exhibited by the isolates. Most of the antagonistic isolates were found to be Gram-negative, motile rods and were oxidase positive, and oxidative in the oxidation and fermentation test, suggesting that they are belong to the genera Pseudomonas . The antagonistic isolates lyzed the dead cells of marine Gram-negative bacteria in both plate and liquid methods. Bacteriolytic and casein hydrolytic activities were observed in the culture supernatant of the isolates. Anion exchange column chromatography (Toyopearl DEAE-650 M) was used to purify the extracellular protease produced by an antagonistic strain A1-J25a. The active fractions of protease collected from the eluted solution also exhibited bacteriolytic activity.     

Water quality and nitrogen budget in turbot Scophthalmus maximus culture system supplemented with lactic acid bacteria

To understand the nutrient utilization efficiency and distribution in indoor fish culture system under treatment of probiotics, this study examined water quality and nitrogen budget in turbot Scophthalmus maximus culture system supplemented with four strains of lactic acid bacteria (LAB) isolated from fishery products. The results showed that the concentration of ammonia nitrogen (NH?‐N) and nitrite nitrogen (NO?‐N) in LAB treatments were all significantly lower (p < 0.05) than in the control group. The nitrate nitrogen (NO?‐N) in LAB treatments showed no significant differences (p > 0.05) compared with control group. The fish feed and water exchange accounted for 79.07% and 17.02% of total N input respectively. For N output, the drainage and residual diets in LAB treatment accounted for 24.50%–25.80% and 1.33%–1.60% respectively, and they were significantly lower than in the control group (27.60% and 2.20% respectively). Fish growth and lost N in LAB treatments accounted for 27.10%–30.50% and 11.00%–18.50% respectively, and they were both significantly higher (p < 0.05) than in the control group (22.30% and 5.30% respectively). The results indicated that the indigenous LAB strains were capable of improving fish growth, and reducing NH?‐N and NO?‐N level (at concentration of 10⁵ cfu/ml) by directly adding in S. maximus culture water. Moreover, specific strains of LAB may increase nitrogen loss by promoting denitrification process in culture system.