Isolation,Screening, and Identification of Proteolytic Lactic Acid Bacteria from Indigenous Chao Product

The aim of the study was to isolate, select, and identify proteolytic lactic acid bacteria (LAB) from chao, a traditional fermented fish from Pangkajene and Kepulauan Regency, South Sulawesi, Indonesia. LAB was isolated by poured plate method. Proteolytic LAB were selected using agar skim milk media. Protease activity of LAB was determined based on the amount of tyrosine released in unit/mL. Proteolytic LAB were identified using API 50 CHL kit and 16S rRNA gene sequence analysis. The result showed that a total of 60 isolates were obtained from chao, and 57% of them were cocci-shape. Fifteen isolates were halotolerant proteolytic LAB. Their R values and protease activity were 2.11–3.39 and 0.267–0.304 U/mL, respectively. Identification by API 50 CHL kit showed that four rod-shape isolates were Lactobacillus plantarum, and two others were Lactobacillus curvatus. Cocci-shape isolates could not be identified as cocci bacterium. Rep-PCR results showed that there were two kinds of bands, namely thick and thin. Two isolates were selected from two types of bands that had the highest R for the analysis of 16S rRNA gene sequences, namely Ags1-3 and Ags7-3. The results showed that Ags1-3 isolate was identified as Lactobacillus plantarum and Ags7-3 as Pediococcus acidilactici.     

Application of Native Lactic Acid Bacteria (LAB) for Fermentative Recovery of Lipids and Proteins from Fish Processing Wastes: Bioactivities of Fermentation Products

Fermentation using native lactic acid bacteria (LAB) was evaluated for its effectiveness in recovering lipids and proteins simultaneously from freshwater fish visceral waste (FVW). Five different LAB isolated from fish processing waste were employed in a fermentation process that involved 10% (w/w) glucose, 2% (w/w) NaCl, and 10% (v/w) LAB. Cultures evaluated included four native isolates (Pediococcus acidilactici NCIM5368, Enterococcus faecalis NCIM5367, Pediococcus acidilactici FM37, and Pediococcus acidilactici MW2) from FVW with E. faecium NCIM5335 as the reference culture. Fermentation with native LAB resulted in recovery of > 90% oil present in the material as against no recovery in case of raw (unfermented) viscera and resulted in > 50% of degree of hydrolysis of proteins. The fatty acid profile of lipids was not affected by the fermentation process. The fermentation liquor, rich in hydrolyzed protein, exhibited antioxidant as well as antagonistic properties against several bacterial pathogens. The results clearly demonstrate the usefulness of fermentation using native isolates for simultaneous recovery of lipids and proteins from fish processing waste. It also asserts the value of fermentation as an eco-friendly method and aids in minimizing disposal/pollution problems associated with these solid wastes.     

Microbial and chemical properties of <Emphasis Type="Italic">aji-no-susu</Emphasis>, a traditional fermented fish with rice product in the Noto Peninsula,Japan

Aji-no-susu is a Japanese fermented fish product prepared from salted horse mackerel Trachurus japonicus, and cooked rice. We studied the organic acid and free amino acid contents and microflora in 12 aji-no-susu products to clarify their features as a lactic-acid-fermented food. Salinity of the samples was approximately 7.0% (rice portion) and 6.0% (fish portion) (w/w). Water activity was approximately 0.9, and pH was approximately 4.4 and lower. In the rice portions, lactic acid content was very high (57 mg/g sample). The predominant amino acids were alanine (2.3 mg/g rice portion) and lysine (2.1 mg/g). In the case of long-fermented (4 and 12 months) aji-no-susu, a high content of gamma-aminobutyric acid (GABA, 1.5 and 1.4 mg/g) was detected. Total viable counts in rice and fish portions were 7.7 and 7.4 log colony-forming units (cfu)/g, respectively. The number of lactobacilli in the rice and fish portions was 7.3 and 7.1 log cfu/g, respectively. Yeasts were detected in eight samples. Furthermore, acid-tolerant lactic acid bacteria (LAB) (Lactobacillus plantarum), GABA-producing LAB (Lactobacillus sp.), and halophilic or halo-tolerant yeast (Debaryomyces hansenii) were isolated and identified. Results in this study indicate that aji-no-susu is a typical traditional lactic-acid-fermented fish product.     

Antimicrobial Activity of 1% Cetylpyridinium Chloride Against Listeria spp. on Fish

Cetylpyridinium chloride (CPC) was evaluated as an antimicrobial against Listeria monocytogenes (LM) and Listeria innocua (LI) on fish. LI and LM showed the same reduction profile and the same minimum inhibitory and bactericidal concentrations. When CPC was applied to a diced fish matrix, reductions of 3.17–3.26 and 3.07–3.19 log were determined for LI and LM, respectively. Antimicrobial activity of CPC against LI resulted in initial reductions of 2.8–3.1 log in fresh fish fillets, 0.9–1.1 log in smoked fish fillets, and 3.2–3.4 log on fish skin. Effectiveness of CPC against total plate counts on fish skin showed reductions of 2.4–2.9 log.     

Biopreservative effect of pediocin ACCEL on refrigerated seafood

To investigate the biopreservative effectiveness of pediocin ACCEL on refrigerated seafoods, fresh fish fillets were immersed in various concentrations of pediocin ACCEL and then stored at either 4° or 0°C. Samples treated with nisin were used as a positive control. The aerobic plate counts (APC) of samples with bacteriocins were <2.0 log₁₀cfu/g (log cfu/g) after 2 days storage at 0°C, except that with 1500 IU/mL of pediocin ACCEL. The APC of samples with nisin were >2.0 log cfu/g after 2 days storage, while those with pediocin ACCEL occurred after 1 day storage at 4°C. In refrigerated seafoods, pediocin ACCEL and nisin suppressed the growth of inoculated Listeria monocytogenes during 2- and 1-week storage at 4°C, respectively. Compared with nisin, the pediocin ACCEL was considered to be more effective on the suppression of L. monocytogenes growth in refrigerated seafoods during 2-week storage at 4°C.     

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.     

Effect of Fish Gelatin Coating Enriched with Oregano Essential Oil on the Quality of Refrigerated Rainbow Trout Fillet

ABSTRACT

In the present study, the effect of fish gelatin coating (FG; 4%, w/v) containing oregano (Origanum vulgare L.) essential oil (OEO; 1.2% w/v) on shelf-life extension of rainbow trout (Oncorhynchus mykiss) fillet stored under refrigerated storage (4 ± 1ºC) was investigated. The control and the treated fish samples were analyzed periodically for chemical (pH; total volatile basic nitrogen, TVB-N; peroxide value, PV; thiobarbituric acid, TBA) and microbiological (total viable count, TVC; psychrotrophic count) characteristics. The FG + OEO treatment produced significantly lower (p < 0.05) TVB-N values as compared to control and FG-coated samples after Day 6 and until the end of the storage period. The TBA values remained low and were found to fluctuate during storage. Results showed that TVC exceeded 6 log CFU g^?1 on Day 8 of storage for control and FG-coated samples and Day 12 for samples coated with FG + OEO. The reduction of 1.05 and 0.83 log CFU g^?1 of psychrotrophic counts was achieved by FG + OEO coating in trout fillet (p < 0.05) as compared with control and FG-coated samples, respectively. Therefore, the incorporation of OEO into FG coating may be a promising method of maintaining the storage quality of the rainbow trout fillet stored at refrigerated temperature.     

Isolation and Antimicrobial Resistance of Staphylococcus spp. in Freshwater Fish and Greek Marketplaces

The presence and antibiotic resistance of Staphylococcus spp. in freshwater fish and the environment of fish markets of Northern Greece were investigated. A total of 269 samples were examined, consisting of 71 rainbow trout (Oncorhynchus mykiss), 65 gibel carp (Carassius gibelio), and 133 environmental samples swabbed from various surfaces at fish markets.Staphylococcus spp. was isolated from 27% of the samples, and 16%, 9%, 0.7%, 0.4% and 0.4% were found to be positive for S. aureus, S. epidermidis, S. warneri, S. hominis-hominis, and S. haemolyticus, respectively. All isolates were examined for their susceptibility to 20 antibiotics. None of the S. aureus isolates were resistant to oxacillin and glycopeptides. Three S. epidermidis, two S. Warneri, and one S. haemolyticus isolates were resistant to oxacillin (methicillin resistant Staphylococcus, MRS). All isolates, except one, showed resistance to as many as 15 antibiotics. The population of Staphylococcus spp. in fish did not exceed 1.0 log CFU/g. Freshwater fish and the environments of retail fish markets were found to harbor multi-drug resistant staphylococci. Whether these findings present a real health risk for humans and to what extent needs to be evaluated.     

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.     

Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.     

The Effects of Fermentation Process with Acid and Lactic Acid Bacteria Strains on the Biogenic Amine Formation of Wet and Spray-Dried Fish Silages of Discards

The impacts of fermentation process with acid and lactic acid bacteria strains (Lactobacillus plantarum, Lactobacillus brevis, Pediococcus acidilactici, Enterococcus gallinarum, and Streptococcus spp.) on the biogenic amine formation of wet and spray-dried fish silage obtained from whole gibel carp (Carassius gibelio, freshwater discard), whole ponyfish (Equulites klunzingeri, seawater discard), and processing by-products of seabass (Dicentrarchus labrax) were investigated. The results showed that among biogenic amines, cadaverine, putrescine, spermidine, spermine, serotonin, dopamine, and agmatine were predominant in all groups. Significant differences (p < 0.05) in biogenic amine concentrations of wet and spray-dried fish silage were observed. Raw fish and wet silages contained histamine level lower than the allowable limit of 50mg/kg, indicating the use of raw fish material with low microbial counts. In addition, no histamine was detected in spray-dried fish silage, except for seabass by-products with a trace quantity of histamine (<0.04mg/100g). The starter culture used for silage preparation did not effectively retard formation of biogenic amines compared to acid silage. It can be concluded that there is potential use of fermented fish silage as a protein source and possibly as a probiotic ingredient for animal feed in both wet and dry form.     

Assessing and enhancing the antimicrobial effect of nisin in soy-seasoned salmon Oncorynchus keta roe using a Pediococcus pentosaceus fermentate and pectin

The compatibility and potential of nisin, a Pediococcus pentosaceus fermentate, and pectin to inhibit Listeria monocytogenes in soy-seasoned salmon roe at 5 and 12°C was studied. The compatibility of nisin and soy-seasoning was assessed by adding 6.3 μg/g of nisin to freshly washed or soy-seasoned (60% soy, 15% sweet-sake, 5% sugar, and 15% water) roe and monitoring the growth of an inoculum of L. monocytogenes (5.6 Log CFU/g; strains IID 578, IID 581, ATCC 7644) as well as residual nisin concentrations at 12°C. The combined antimicrobial potential was determined by monitoring growth of the inoculum (5.2 Log CFU/g) in roe when the soy-seasoning contained nisin (0.05 g/ml), fermentate powder made by freeze-drying a broth culture of P. pentosaceus (0.1 g/ml), and 2% pectin at 5 or 12°C. Chloride content, total acid content, and the effect of pectin on acceptability of roe were also determined. Nisin was more effective in soy-seasoned roe than in freshly washed roe. Microbial growth was completely inhibited by a combination of soy-seasoning, nisin, the fermentate, and pectin. Despite its viscosity, pectin did not significantly affect the chemical properties or acceptability of roe relative to pectin-free roe samples (P < 0.05). Based on these results, we conclude that nisin’s potential as an antimicrobial agent in soy-seasoned roe can be enhanced using fermentates and pectin.     

Differential pathogenicity of five Streptococcus agalactiae isolates of diverse geographic origin in Nile tilapia (Oreochromis niloticus L.)

Streptococcus agalactiae is an emerging pathogen of fish and has caused significant morbidity and mortality worldwide. The main objective of this study is to assess whether pathogenic differences exist among isolates from different geographic locations. Nile tilapia (Oreochromis niloticus L.) were administered an intraperitoneal injection of suspension containing USA, Brazil, Honduras, Israel, or Kuwait S. agalactiae isolates at concentrations ranging from 10² to 10⁷ cfu mL^?1. The LD₅₀ values 7 days after challenge were as follows: USA (1.0 × 10² cfu mL^?1), Brazil (1.5 × 10³ cfu mL^?1), Honduras (6.8 × 10³ cfu mL^?1), Israel (1.0 × 10⁴ cfu mL^?1) and Kuwait (7.2 × 10⁵ cfu mL^?1). Fish from all groups exhibited lethargy, anorexia, exophthalmia, corneal opacity, erratic swimming, petechiae and mortality. Opercular clearing and ascites were only found after infection with certain geographic isolates. The findings in this study indicate that S. agalactiae isolates of different geographic origin can cause significant mortalities after experimental challenge and can have different pathogenic capacities. Isolates from the Americas (USA, Brazil and Honduras) were more pathogenic to Nile tilapia than isolates from the Middle East/Asia (Israel and Kuwait).     

Virulence of Flavobacterium psychrophilum isolates in rainbow trout Oncorhynchus mykiss (Walbaum)

Flavobacterium psychrophilum, the causative agent of bacterial cold‐water disease (BCWD) in freshwater‐reared salmonids, is also a common commensal organism of healthy fish. The virulence potential of F. psychrophilum isolates obtained from BCWD cases in Ontario between 1994 and 2009 was evaluated. In preliminary infection trials of rainbow trout juveniles, significant differences (0% to 63% mortality) in the virulence of the 22 isolates tested were noted following intraperitoneal injection with 10⁸ cfu/fish. A highly virulent strain, FPG 101, was selected for further study. When fish were injected intraperitoneally with a 10⁶, 10⁷ or 10⁸ cfu/fish of F. psychrophilum FPG 101, the 10⁸ cfu/fish dose produced significantly greater mortality (p < 0.05). The bacterial load in spleen samples collected from fish every 3 days after infection was determined using rpoC quantitative polymerase chain reaction amplification and by plate counting. Bacterial culture and rpoC qPCR were highly correlated (R² = 0.92); however, culture was more sensitive than the qPCR assay for the detection of F. psychrophilum in spleen tissue. Ninety‐seven per cent of the asymptomatic and the morbid fish had splenic bacterial loads of <2.8 log₁₀ gene/copies and >3.0 log₁₀ gene copies/reaction, respectively, following infection with 10⁸ cfu/fish.     

Colonization and probiotic effects of lactic acid bacteria in the gut of the abalone Haliotis gigantea

Strains of lactic acid bacteria (LAB) were isolated from several different sources and evaluated in vitro for potential probiotic effects in abalones. Two isolates (Lactobacillus sp. strain a3 and Enterococcus sp. strain s6) were highly resistant to bile salt and/or gastric juice and inhibited the growth of three abalone pathogens (Listonella anguillarum, Vibrio harveyi, and V. carchariae). Each of the LAB isolates was used to supplement diet of the abalone Haliotis gigantea for a period of 3 weeks. One group of animals received Lactobacillus sp. strain a3 added to commercial dry feed, one group received Enterococcus sp. strain s6 added to the feed, and a control group received only standard commercial feed. Culturable LAB counts of gut homogenates indicate the a3 colonized in the gut of abalones. Digestive enzyme activities and the concentrations of a number of volatile short-chain fatty acids (VSCFA) were elevated in the gut of abalones receiving feed supplemented with the two LAB strains. These results indicate that dietary supplementation can enable LAB colonization or persistence in the gut of abalone species and can potentially enhance probiotic effects.     

Correlation of Kudoa Spore Counts with Proteolytic Activity and Texture of Fish Mince from Pacific Hake (Merluccius productus)

ABSTRACT

Sixteen batches of fish mince prepared using fillets from Pacific hake (Merluccius productus) harvested near Vancouver Island, Canada were studied for possible correlations of Kudoa spore counts with proteolytic activity and cooked texture. The fish were mainly infected by K. paniformis, and Kudoa spores were distributed throughout 7 sampling sites on 13 individual hake fish tested. Proteolytic activity of fish homogenate was optimum within the range of pH 5.25–5.50 and 52–55°C. Significant (p < 0.05) correlations were observed among fish mince K. paniformis spore counts, endogenous proteolytic activity, cooked texture measured as maximum compression force (g), and the change in free amino groups during autolysis.     

Roles of surface layer proteins in the regulation of Pediococcus pentosaceus on growth performance,intestinal microbiota,and resistance to Aeromonas hydrophila in the freshwater prawn Macrobrachium rosenbergii

Maintaining the balance of intestinal microbiota is vital for the growth and health of crustaceans. The current study explored the influences of the surface layer proteins (SLPs) of Pediococcus pentosaceus on the growth performance, disease resistance, and gut microbial community of the freshwater prawn Macrobrachium rosenbergii. After 28 days of feeding, obvious enhancements in the growth performance were found in the PF (a commercial basic diet + intact P. pentosaceus) group compared to the CF (a commercial basic diet, control) and RF (a commercial basic diet + LiCl-treated P. pentosaceus) groups. Meanwhile, the relative percent survival (RPS) of the PF group was found to be 82.1%, higher than the RF group (RPS = 63.2%) in the challenge test. There were noteworthy differences in the relative richness of Aeromonas among different treatments, and the PF group was the lowest. Whereas the relative abundance of Lactococcus between the CF and RF group did not markedly differences, which was obviously lower than the PF group. The prediction of bacterial phenotype indicated that the pathogenic potential of intestinal microbiota was decreased by increasing dietary SLPs-carrying P. pentosaceus. The results suggested that SLPs-carrying P. pentosaceus can improve growth, modulate intestinal microbiota, and increase the resistance of prawns against Aeromonas hydrophila infection.

     

Sensory and Microbial Quality of Retail Finfish With Emphasis on Antibiotic-Resistant Bacteria

The sensory and microbial quality of commercially important retail finfish of Kolkata, India was evaluated emphasizing oxytetracycline and chloramphenicol resistant bacterial contamination. The finfish had total viable counts (TVCs) in the range of log 4.30/g in Labeo bata to log 7.93/g in Mystus tengara. The counts of oxytetracycline and chloramphenicol resistant bacteria were in the range of log 2.00–6.79/g and log 1.70–5.80/g fish, respectively. About 72% of the Kolkata market finfish harbored viable bacteria exceeding the acceptable international limit of 5.0?×?10⁵/g. Up to 3.45 and 29.86% of TVCs were resistant to chloramphenicol and oxytetracycline, respectively. The marine fish sold in the Kolkata market were of marginal to inferior quality with the highest mean quality index point (QIP) range of 6.80–10.20. Most freshwater fish were of fresh to marginal quality and recorded QIP in the range of 3.80–8.20. Though the freshwater fish were judged fresh by the quality index method in comparison to the marine counterparts, they recorded high viable counts. The results of the present study call for the need to educate the fish handlers on appropriate hygiene and safe handling of fish and the public about fish-borne bacterial diseases and the impact of antibiotic resistant bacteria on human health. They also show the potential for fish to be a source of antibiotic bacteria for human consumers.     

冰鲜大黄鱼加工过程中的细菌学分析及关键控制点

探讨了冰鲜大黄鱼加工过程中鱼体温度变化及细菌总数和主要腐败菌假单胞菌的变化,分析了加工中的关键控制点。活鱼、加工结束后中心温度超过8℃的鱼的细菌总数、假单胞菌数分别为5.5×104、4.5×104cfu/g和3.3×104、1.4×103cfu/g;加工结束后中心温度未超过8℃的鱼,细菌总数和假单胞菌数分别为9.6×103、3.8×103cfu/g;冰藏2d、5d后鱼的细菌总数、假单胞菌数分别为1.1×104、8.6×103cfu/g和2.4×103、5.6×103cfu/g。并从细菌学角度确定了原料鱼、冰、预冷却、运输、加工是冰鲜大黄鱼加工过程中的关键控制点。     

Microbiological Quality of Ready-to-Eat Pickled Fish Products

The microbiological quality of 18 commercially available in Spain ready-to-eat fish products containing Engraulidae was evaluated through application of the corresponding ISO procedures for total mesophilic aerobic microbial counts, detection and enumeration of enterobacteria, and detection of Staphylococcus spp. All isolates were identified to the species level using two different biochemical methods: the API® test and the Biolog® identification system. The most commonly occurring contaminants found were Enterobacteriaceae—such as Citrobacter freundii and other Citrobacter species, Enterobacter cloacae, Cronobacter sakazakii, Hafnia alvei, Pantoea, Proteus ssp., and Escherichia coli. The presence of such opportunistic pathogens and contaminant microflora was confirmed in 61% of the foods sampled.