Evaluation of probiotic characteristics and whole genome analysis of Pediococcus pentosaceus MR001 for use as probiotic bacteria in shrimp aquaculture.

The development of non-antibiotic and environmentally friendly agents is a key consideration for health management in shrimp aquaculture. In this study, the probiotic potential in shrimp aquaculture of Pediococcus pentosaceus MR001, isolated from Macrobrachium rosenbergii, was investigated by means of feeding trial and genetic characterization. In the feeding trial, dietary supplementation with P. pentosaceus MR001 significantly increased weight gain and digestive enzyme activity (p < 0.05) in shrimp, Litopenaeus vannamei. The intestinal histology showed that shrimp given the probiotic diet had healthier guts than the control group. Also, the immune gene expression and the survival rate in the treatment group were significantly increased when compared with the control group. The genetic characteristics of P. pentosaceus strain MR001 were explored by performing whole-genome sequencing (WGS) using the HiSeq 2500 platform and PacBio system, revealing the complete circular genome of 1,804,896 bp. We also identified 1789 coding genes and subsequently characterized genes related to the biosynthesis of bacteriocins, stress resistance, and bile tolerance. Our findings suggest that insights in the functional and genetic characteristics of P. pentosaceus strain MR001 could provide opportunities for applications of such strain in shrimp diet supplementation.

Detección de protozoos parásitos entéricos, bacteriófagos de E. coli y organismos indicadores de contaminación en camarones comercializados en el estado Zulia / Detection of enteric protozoa parasites, E. coli bacteriophages and fecal pollution Indicator organisms in shrimp sold in the state of Zulia

Se determinó la presencia de Giardia intestinalis y Cryptospodidium parvum, bacteriófagos de Escherichia coli y organismos indicadores de contaminación (OIC), en muestras de camarones para el consumo humano comercializados en el estado Zulia. Los parásitos se concentraron a partir de sistemas digestivos de pools de camarones por la técnica de formol-éter y se cuantificaron por inmunofluorescencia directa. La concentración de los bacteriófagos de E. coli F+ y los OIC se evaluó por técnicas estándar. En este trabajo se detectó la presencia de G. intestinalis, C. parvum, bacteriófagos y E. coli en camarones comercializados en el estado Zulia que cumplían los criterios de la normativa venezolana de calidad sanitaria e inocuidad. Del total de muestras analizadas el 91,5% fueron positivas para G. intestinalis (promedio: 36,6 quistes/100g), 95,3% para C. parvum (promedio: 32,8 ooquistes/100g), 100% para los bacteriófagos de E coli F+ (promedio de 2,8 x 103 UFP/100 g) y 71,5% para E. coli (promedio de 4,3 x 104 NMP/g). Los resultados obtenidos indican que los camarones pueden convertirse en un vehículo para la transmisión de patógenos al hombre y dejan en evidencia la necesidad de la inclusión de un parámetro parasitológico y viral en el control de la calidad microbiológica de estos productos alimenticios.

The presence of G. intestinalis and C. parvum, E. coli F+ bacteriophages and fecal pollution indicator organisms was determined in shrimp for human consumption marketed in the State of Zulia. Parasites were concentrated from the digestive systems of shrimp pools, detected by formalin-ether and quantified by direct immunofluorescence. E. coli F + bacteriophage and pollution indicator organism concentrations were determined by standard techniques. In this work, G. intestinalis, C. parvum, E. coli F + bacteriophages and E. coli were detected in shrimp for human consumption marketed in the State of Zulia that met the quality criteria of Venezuelan health and safety regulations. 91.5% of the samples analyzed were positive for G. intestinalis (average: 36.6 cyst/100g), 95.3% for C parvum (average: 32.8 oocyst/100g), 100% for E coli F + bacteriophages (average: 2.8 x 103 FPFU/100g) and 71.5% for E. coli (average: 4.3 x 104 MPN/g). Results of this research indicate that shrimp can become a vehicle for transmitting pathogens to humans and demonstrate the need for including a parasitic and viral parameter in microbiological quality control for seafood.

Effect of brine marination on survival and growth of spoilage and pathogenic bacteria during processing and subsequent storage of ready-to-eat shrimp (Pandalus borealis).

The effect of brine marination at chill temperatures on survival and growth of spoilage and pathogenic bacteria during processing and subsequent storage of ready-to-eat cold water shrimp was studied. Survival and growth of Lactobacillus sakei, Listeria monocytogenes, Salmonella, Staphylococcus aureus and Vibrio parahaemolyticus were examined. The effect of brine composition and pH was determined in 12 screening experiments without addition of shrimp. Sixteen challenge tests with shrimp were then carried out to examine the effect of brine composition and storage temperature on survival and growth during processing and subsequent storage of brined and drained shrimp in modified atmosphere packaging (MAP). Different brines with (i) acetic and lactic acids (AL) or (ii) benzoic, citric and sorbic acids (BCS) were studied. V. parahaemolyticus was inactivated in brine AL without shrimp whereas concentrations of all the examined microorganisms were reduced in brine BCS. A significant effect of brine pH on inactivation was observed and inactivation during chill marination of shrimp in brine was reduced compared to the effect of brine alone. This was explained by a relatively fast increase of pH in the brine during marination of shrimp. For shrimp in brine BCS, reductions were observed for V. parahaemolyticus and Salmonella, whereas inactivation in shrimp was only noticed for Salmonella in brine AL. The observed reductions were too small to be used in practise for decontamination of shellfish. None of the examined pathogens was able to grow at 7°C in brined and drained MAP shrimp that resembled commercial products. However, reducing the concentration of acetic and lactic acids by 50% resulted in relatively fast growth of L. monocytogenes in brined and drained MAP shrimp at 7°C. Growth of S. aureus and Salmonella was observed in similar products stored at 15°C. V. parahaemolyticus was reduced in brined and drained MAP shrimp stored at both 7 and 15°C. Based on the results of the present study, L. monocytogenes was identified as the greatest potential risk with respect to the safety of brined and drained MAP shrimp. The potential of L. sakei as spoilage bacterium in brined and drained MAP shrimp was confirmed. Importantly, growth rates of L. sakei and L. monocytogenes in brined and drained MAP shrimp were predicted accurately by available mathematical models. Thus, these models can be used for product development and establishment of shell-life for ready-to-eat shrimp taking into account both quality and safety aspects.

Occurrence and activity of microorganisms in shrimp waste.

This study aimed to determine the occurrence and respiration activity of heterotrophic bacteria and fungi in shrimp shell waste and to evaluate the role of chitinolytic bacteria and fungi in its decomposition. The highest levels of bacteria were found in shrimp heads sections and the lowest in exoskeletons. The level of fungi was much lower, with the highest proportion present in heads sections and the lowest in exoskeletons. Chitinolytic bacteria constituted a small percentage of the total heterotrophic bacteria in fresh shrimp waste, averaging 4% in exoskeletons, 2.4% in all parts, and 2% in heads. No chitinolytic bacteria were detected in stored waste. In contrast, the percentage of chitinolytic fungi in shrimp waste was much higher than that of bacteria. Chitinolytic fungi constituted 25-60% of the total fungi in fresh waste and 15-40% in stored waste. Chitinolytic bacteria isolated from heads sections were characterized by the highest chitinolytic activity, averaging 11.2 nmol of methylumbelliferyl x mg(-1) protein x h(-1), whereas the lowest activity was in strains from exoskeletons, averaging 3.2 nmol of methylumbelliferyl x mg(-1) protein x h(-1). The chitinolytic activity of fungi isolated from all parts waste, head sections, and exoskeletons was similar. The respiration activity of microorganisms in fresh and stored waste was similar. Oxygen consumption activity increased during incubation and approached a saturation value between days 4 and 5. No correlation between the end value of respiratory activity in the analyzed section of shrimp discard after 5 days and the level of bacteria and fungi was observed. The only significant correlation observed was between the respiratory activity of the shrimp and the level of fungi. The respiration activity significantly depended on the analyzed section of shrimp discard (p<0.000).

Microbial changes and growth of Listeria monocytogenes during chilled storage of brined shrimp (Pandalus borealis).

Thirteen storage trials and ten challenge tests were carried out to examine microbial changes, spoilage and the potential growth of Listeria monocytogenes in brined shrimp (Pandalus borealis). Shrimp in brine as well as brined and drained shrimp in modified atmosphere packaging (MAP) were produced and studied. Different recipes were used to study the effect of preserving parameters (organic acids, pH and NaCl) on growth of microorganisms and shelf life at 7-8 degrees C or 12 degrees C. Particularly, brines with different concentrations of (i) benzoic, citric and sorbic acids or (ii) acetic, citric and lactic acids were studied. Furthermore, the effect of adding diacetate to brined shrimp was evaluated. A single batch of cooked and peeled shrimp was used to study both industrially and manually processed brined shrimp with respect to the effect of process hygiene on microbial changes and the shelf life of products. Concentrations of microorganisms on newly produced brined shrimp from an industrial scale processing line were 1.0-2.3 log (CFU g(-1)) higher than comparable concentrations in manually processed samples. This resulted in a substantially shorter shelf life and a more diverse spoilage microflora of the industrially processed brined shrimp. In addition, shelf life of brined shrimp was affected by the types and concentrations of organic acids and by the storage temperature as expected. The effect of MAP was less pronounced. Eighty-two isolates from the spoilage microflora of brined shrimp were identified and they included 53 lactic acid bacteria, 6 coagulase negative Staphylococcus spp., 18 Pseudomonas fluorescens and 5 yeast isolates. After storage at 7 degrees C, P. fluorescens, Enterococcus-like isolates, E. malodoratus, Carnobacterium maltaromaticum, coagulase negative Staphylococcus spp. and Lactobacillus sakei constituted the dominating microflora of shrimp in brines that contained benzoic, citric and sorbic acids as preservatives. L. sakei dominated the spoilage microflora of brined and drained MAP shrimp, and of brined shrimp preserved using acetic, citric and lactic acids, irrespective of packaging conditions. Shrimp in brine with benzoic, citric and sorbic acids prevented growth of L. monocytogenes during more than 40 days at 7 degrees C when the preserving parameters resembled those of commercial products. However, small changes in the preserving parameters and, particularly, reduced concentrations of benzoic acid led to growth of L. monocytogenes in brined shrimp. The present study provides significant new information on microbial changes, shelf life and growth of L. monocytogenes in brined shrimp. This information can facilitate development of new and safe brined shrimp products.

Halalkalicoccus jeotgali sp. nov., a halophilic archaeon from shrimp jeotgal, a traditional Korean fermented seafood.

A novel, extremely halophilic archaeon B3(T) was isolated from shrimp-salted seafood. Its morphology, physiology, biochemical features and 16S rRNA gene sequence were characterized. Strain B3(T) is non-motile, Gram-variable, requires at least 10 % (w/v) NaCl for growth and grows in the ranges of 21-50 degrees C and pH 6.5-9.0. The DNA G+C content of strain B3(T) was 63.2 mol%. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain B3(T) belonged to the genus Halalkalicoccus and was phylogenetically closely related to the type strain Halalkalicoccus tibetensis (98.64 %). However, DNA-DNA hybridization experiments showed 7.0 % relatedness between strain B3(T) and a strain of a reference species of the genus Halalkalicoccus. Combined analysis of 16S rRNA gene sequences, DNA-DNA relatedness data, physiological and biochemical tests indicated that the genotypic and phenotypic characteristics differentiate strain B3(T) from other Halalkalicoccus species. On the basis of the evidence presented in this report, strain B3(T) represents a novel species of the genus Halalkalicoccus, for which the name Halalkalicoccus jeotgali. sp. nov. is proposed. The type strain is B3(T) (=KCTC 4019(T)=DSM 18796(T)=JCM 14584(T)=CECT 7217(T)).

Salinicoccus siamensis sp. nov., isolated from fermented shrimp paste in Thailand.

Fifteen strains of moderately halophilic, Gram-positive cocci were isolated from a traditional fermented shrimp paste ('ka-pi') produced in Thailand. These bacteria were strictly aerobic, non-motile, non-sporulating and catalase- and oxidase-positive. They produced orange pigment and grew in the presence of 1.5-25 % (w/v) NaCl. They grew optimally in 10 % (w/v) NaCl, at pH 8.5 and at 37 degrees C. The cell-wall peptidoglycan was of l-Lys type. Menaquinone with six isoprene units (MK-6) was a major component. The dominant cellular fatty acids were anteiso-C(15 : 0) and iso-C(15 : 0). DNA G+C contents were in the range 44.5-47.5 mol%. Comparative 16S rRNA gene sequence analyses indicated that representative strain PN1-2(T) was related most closely to Salinicoccus roseus JCM 14630(T), with 97.3 % similarity. The other novel strains were included in the same species based on their levels of DNA-DNA relatedness to strain PN1-2(T) (> or =76.6 %) but showed low DNA-DNA relatedness to S. roseus JCM 14630(T) (21.7 %). On the basis of the phenotypic and molecular data presented, the 15 novel strains are suggested to represent a single novel species of the genus Salinicoccus, for which the name Salinicoccus siamensis sp. nov. is proposed. The type strain is PN1-2(T) (=JCM 12822(T) =PCU 242(T) =TISTR 1562(T)).

Natronococcus jeotgali sp. nov., a halophilic archaeon isolated from shrimp jeotgal, a traditional fermented seafood from Korea.

A novel halophilic archaeon (strain B1(T)) belonging to the genus Natronococcus was isolated from shrimp jeotgal, a traditional fermented food from Korea. Colonies of this strain were orange-red and cells were non-motile cocci that stained Gram-variable. Strain B1(T) grew in 7.5-30.0 % (w/v) NaCl and at 21-50 degrees C and pH 7.0-9.5, with optimal growth occurring in 23-25 % (w/v) NaCl and at 37-45 degrees C and pH 7.5. Strain B1(T) was most closely related to the type strain of Natronococcus occultus, with which it shared 97.91 % 16S rRNA gene sequence similarity. Within the phylogenetic tree, this novel strain shared a branching point with N. occultus and occupied a phylogenetic position that was distinct from the main Natronococcus branch. The degree of DNA-DNA hybridization with the type strain of N. occultus, the most closely related species phylogenetically, was 16.4 %. On the basis of these results, it is concluded that strain B1(T) represents a novel species of the genus Natronococcus, for which the name Natronococcus jeotgali is proposed. The type strain is B1(T) (=KCTC 4018(T)=DSM 18795(T)=JCM 14583(T)=CECT 7216(T)).

Contamination of cooked peeled shrimp (Pandalus borealis) by Listeria monocytogenes during processing at two processing plants.

Listeria spp. and Listeria monocytogenes contamination was evaluated in cooked peeled shrimp (final or semifinal product, 82 samples) and the shrimp-processing environment (two plants, 613 samples) in eight surveys conducted from 1998 through 2001. Listeria was detected in 12.5% (78) of the 695 samples (11.2% of the samples were positive for L. monocytogenes), but none of the samples of final product contained Listeria. One hundred seventy-two L. monocytogenes isolates were characterized by pulsed-field gel electrophoresis. Cleavage with macrorestriction enzymes AscI and ApaI yielded 14 different pulsotypes in the plants; two types were dominant, one in each plant. Sixty-three of the 106 isolates in plant A and 43 of the 66 isolates in plant B were of the dominant types. Certain strains, mainly of serotypes 1/2c and 4b and pulsotypes 1A and 2H, were persistent for long periods in both plants. Adaptation of good hygienic practices in the processing plants, including strict rules concerning traffic of staff and equipment, and existing hygienic requirements appeared to be effective in preventing contamination between areas within plants and in the final product. The persistence of Listeria strains in these two processing plants indicates the importance of detecting the places in the processing environment (e.g., transporters, equipment, floors, and drains) where L. monocytogenes can survive so that cleaning and disinfection efforts can be directed to such niches.

Shelf life and safety aspects of chilled cooked and peeled shrimps (Pandalus borealis) in modified atmosphere packaging.

AIMS: To evaluate the growth of Listeria monocytogenes and shelf life of cooked and peeled shrimps in modified atmosphere packaging (MAP). METHODS AND RESULTS: Storage trials with naturally contaminated cooked and peeled MAP shrimps (Pandalus borealis) were carried out at 2, 5 and 8 degrees C. Challenge tests at the same conditions were performed after inoculation with Listeria monocytogenes. Both storage trials and challenge tests were repeated after 4 months of frozen storage (-22 degrees C). Brochothrix thermosphacta and Carnobacterium maltaromaticum were responsible for sensory spoilage of cooked and peeled MAP shrimps. In challenge tests, growth of L. monocytogenes was observed at all of the storage temperatures studied. At 5 and 8 degrees C the concentration of L. monocytogenes increased more than a 1000-fold before the product became sensory spoiled whereas this was not observed at 2 degrees C. Frozen storage had only a minor inhibiting effect on growth of L. monocytogenes in the thawed product. CONCLUSIONS: To prevent L. monocytogenes becoming a safety problem, cooked and peeled MAP shrimps should be distributed at 2 degrees C and with a maximum shelf life of 20-21 d. At higher temperatures shelf life is significantly reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: Information is provided to establish shelf life of cooked and peeled MAP shrimps.

Microbiological quality of Icelandic cooked-peeled shrimp (Pandalus borealis).

Iceland is a major producer of cold water shrimp, Pandalus borealis. In recent years considerable attention has been given to improve hygiene in the factories producing cooked, peeled and frozen shrimp. To keep track of the bacteriological status of the end product, shrimp from most of the factories is routinely analysed bacteriologically by the request of shrimp exporters. This paper reports on the results of a bacteriological analysis of 7913 samples of shrimp from 26 Icelandic factories over a 6-year period. The results showed that the geometric mean of APC (at 35 degrees C) was 1718 per g and 57% of the samples had APC under 1000 per g. Some 70% of the samples had less than one coliform per g and 99.9% of the samples had less than one faecal coliform per g. Staphylococcus aureus was detected in less than 0.2% of the samples. The results show improvement in bacterial profiles, mainly total coliforms, over the 6-year period. Overall, the results show acceptable bacterial numbers in the finished product, indicating a high level of hygiene. Listeria spp. were, however, found in 270 of 3331 samples examined or 8.1%. Species identification was done on 49 of the 270 positive samples. The proportion of L. monocytogenes was found to be 26.5%.

[Quality changes in iced shrimps (Pandalus borealis). III. Indole and pH in shrimps caught in the Barents Sea compared with shrimps caught in the Far East (author's transl)]. / Kvalitetsforandringer i isete dypvannsreker (Pandalus borealis) III. Indol of pH i reker fanget i Barentshavet sammenlignet med reker fanget i Det fjerne Osten.

The content of indole and the pH have been determined post mortem in shrimps (Pandalus borealis) caught in the Barents Sea and in shrimps caught outside Malaysia, India and Taiwan. These two criteria were compared with organoleptic assessment and the contents of volatile nitrogen bases (ammonia, trimethylamine) and living bacteria. For shrimps caught in the Barents Sea, both raw shrimps stored in ice and processed (broiled, peeled and single-frozen) shrimps were investigated. The results showed that only low levels of indole had been formed during ice-storage. Not until an advanced state of spoilage could a distinct increase in the indole content in raw and in boiled, peeled shrimps be discerned. pH increased slowly and varied in the area between acceptable and not acceptable quality. Neither the indole content nor the pH seems therefore to be a useful criterion for quality assessment either of raw shrimps caught in the Barents Sea or of such shrimps after processing (boiling and peeling). Most of the samples of boiled, peeled shrimps from the Far East were assessed organoleptically as less good-spoiled, and bacterial growth was significant. The content of trimethylamine oxide and volatile nitrogen was low, while the content of indole was high and exceeded 25 microgram/100 g in 8 or 14 samples. This is the upper limit for import in USA. The content of indole seems to be an important quality criterion for shrimps caught in warmer countries. The content of indole exceeded 25 microgram/100 g in some samples which were assessed organoleptically as acceptable. The pH was lower in brine-treated shrimps than in the others.

Microbiological characteristics of Pacific shrimp (Pandalus jordani).

Microorganisms associated with Pacific shrimp (Pandalus jordani) were isolated and identified. Those on the iced raw shrimp, which yielded an average count of 1.6 x 10(6), were predominantly Moraxella, Pseudomonas, Acinetobacter, Arthrobacter, and Flavobacterium-Cytophaga spp. The blanching and peeling reduced the microbial level to 3.3 x 10(4) and also selectively eliminated Moraxella spp. The microbial flora changed after each processing sequence, and the heat sensitivity and growth characteristics of the representative microbial groups suggested that the presence of Arthrobacter and Acinetobacter spp. in peeled shrimp may indicate inadequate cleaning of raw shrimp or a shorter blanching time. The presence of Moraxella and Flavobacterium-Cytophaga spp. would indicate the degree of secondary contamination, and the presence of Pseudomonas spp. would indicate the shelf-age of the processed shrimp.