Molecular and biotechnological characteristics of proteolytic activity from Streptococcus thermophilus as a proteolytic lactic acid bacteria to enhance protein-derived bioactive peptides.

The demand for healthy food items with a high nutrient value of bioavailability and bioaccessibility has created a need for continuous development of technology and food ingredients like bioactive peptides. This study aimed to investigate seven proteolytic lactic acid bacteria (PLABs) isolated from the plaa-som (fermented fish) sample originated from silver BARB species for production of proteolytic enzymes. Proteolytic enzymes produced by (PLABs) were used further to create potent bioactive peptides by hydrolyzing proteins throughout PLAB-probiotics enhancer. Protein derived-bioactive peptides was tested the proteolytic activity on different protein sources and examined bioactivities including antioxidative and antimicrobial effect for further use in functional foods. Results of screened-PLAB strains showed high proteolytic activity namely Streptococcus thermophilus strains (KKUPA22 and KKUPK13). These strains have proteolytic system consisting of extracellular and cell-bound enzymes that used for degrading protein in fish flesh protein (FFP) and skim milk (SKM) broth media. Proteolytic activity of tested bacterial enzymes was estimated after incubation at 45, 37, and 50 °C. Furthermore, FFP hydrolysates were formed with various peptides and has small molecular weights (checked by SDS-PAGE) in the range of10.5 to 22 kDa), exhibiting strong activity. Data revealed that S. thermophilus strains (KKUPA22 and KKUPK13) had high antioxidant activity in term of 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) radical-scavenging inhibition, and ferric reducing antioxidant power (FRAP) reducing power capacity. Both strains (KKUPA22 and KKUPK13) of S. thermophilus have higher antimicrobial activity against Gram-negative bacteria than against Gram-positive bacteria. We have confirmed presence of proteolytic (prt) gene regions in S. thermophilus strains using specific primers via PCR amplification. Results showed highest homology (100%) with the prtS gene of S. thermophillus located on the cell envelope proteolytic enzymes (CEPEs) such as serine proteinase. Therefore, it concluded that the proteolytic system of tested PLAB strains able to generate bioactive peptides-derived proteins having active biological property, good mechanism of degradability, and bioaccessibility for further use in catalyzing protein of functional foods.

Diversity of fermentative yeasts with probiotic potential isolated from Thai fermented food products.

This research aimed to evaluate the diversity of yeasts recovered from fermented foods gathered from some areas of Northeastern Thailand. The fermented food items included Pla-som, Nham-pla, Kem-buknud, Isan-sausage, Pla-ra, Mhum-neu, Mhum-Khai-pla, Nham-neu, Nham-mu, Kung-joom, Som-pla-noi, and Poo-dong. Their probiotic characteristics were also investigated. A total of 103 yeast isolates of nine genera were identified using 28S rDNA sequencing. The yeast genera were Candida (20.3%), Diutina (2.9%), Filobasidium (1.0%), Kazachstania (33.0%), Pichia (3.9%), Saccharomyces (1.0%), Starmerella (28.2%), Torulaspora (2.9%), and Yarrowia (6.8%). Based on probiotic characteristic analysis of ten selected yeast strains, Kazachstania bulderi KKKS4-1 showed the strongest probiotic characteristics in terms of hemolytic activity, antimicrobial activity against pathogenic bacteria, tolerance to low pH and bile salt and hydrophobicity. Isolated yeasts with probiotic characteristics may be useful in fermented food and animal feed production to improve their nutritional values.

Use of Bacteriophage to Control Experimental Aeromonas hydrophila Infection in Tilapia (Oreochromis niloticus).

BACKGROUND AND OBJECTIVE: Antibiotics have been used to treat Aeromonas hydrophila infections in fish farming. However, their extensive uses can cause many negative effects including the development of drug-resistant bacterial strains. The main objective of this study was to find an alternative to antibiotics to inhibit A. hydrophila both in vitro and in vivo. MATERIALS AND METHODS: A bacteriophage infecting A. hydrophila was isolated from a fish a pond water sample. It was classified based on its genome type studied by enzymatic digestion and morphology investigated by transmission electron microscopy. Its ability to control experimental A. hydrophila infection in tilapia (Oreochromis niloticus) was examined by feeding tilapia with fish diets supplemented with different titers of the bacteriophage. RESULTS: A bacteriophage specific to Aeromonas hydrophila UR1 designated PAh4 was isolated and classified as a member of the family Myoviridae. When tilapia experimentally infected with A. hydrophila at the median lethal dose (3.16×105 CFU per fish) were fed the fish diets supplemented with the bacteriophage PAh4 at doses ranging from 105-108 PFU g-1 of diet, the diets could reduce the mortality rate of infected tilapia in a dose-dependent manner. CONCLUSION: The bacteriophage PAh4 can be used as an alternative to antibiotics to control A. hydrophila infection in tilapia.

Effect of Lactobacillus plantarum C014 on innate immune response and disease resistance against Aeromonas hydrophila in hybrid catfish.

A bacterial strain isolated from intestines of hybrid catfish (Clarias gariepinus Male × Clarias macrocephalus Female) exhibited an in vitro inhibitory effect on a fish pathogen, Aeromonas hydrophila TISTR 1321. By using the 16S rDNA sequence analysis, it was identified as Lactobacillus plantarum C014. To examine whether L. plantarum C014 had potential for use as an immunostimulant and biocontrol agent in hybrid catfish, the fish diet supplemented with L. plantarum C014 (10(7) CFU/g diet) was prepared and used for the in vivo investigation of its effect on innate immune response and disease resistance of hybrid catfish. Two innate immune response parameters, phagocytic activity of blood leukocytes and plasma lysozyme activity, were significantly enhanced in the treated fish after 45 days of feeding. Feeding the fish with the L. plantarum C014 supplemented diet for 45 days before challenging them with A. hydrophila at the dose of LD50 could reduce the mortality rate of the fish from 50% (in control group) to 0% (in treated group). Based on its origin and beneficial effect on innate immune response and disease resistance, L. plantarum C014 may be a potential candidate for use as a natural and safe immunostimulant and biocontrol agent in hybrid catfish.

Construction of a food-grade cloning vector for Lactobacillus plantarum and its utilization in a food model.

The development of Lactobacillus plantarum to be used in starter cultures in the food industry has been limited because of the lack of a food-grade cloning vector for the bacterium. In this study, the plasmid pFLP1 was constructed by joining 2 DNA fragments derived from food-approved organisms. The 5.2-kb BamHI/KpnI DNA fragment of pRV566 containing the theta-type replicon of Lactobacillus sakei was ligated to the BamHI/KpnI DNA fragment of a 2.9-kb lactococcal cadmium resistance determinant amplified from pND918. The 8.1-kb newly constructed plasmid could transform L. plantarum N014, a bacteriocin-producing bacteria originally isolated from nham, a traditional Thai fermented sausage. The resulting transformant, L. plantarum N014-FLP, and its parent strain were shown to be very similar in growth rate and bacteriocin activity. In addition, the plasmid was very stable in its host bacteria under nonselective pressure for 100 generations in MRS medium and for 5 days in a nham model. These results suggest that pFLP1 is a potential food-grade cloning vector for L. plantarum.

Identification of viable Listeria species based on reverse transcription-multiplex PCR (RT-MPCR) and restriction digestion.

A novel method for the identification of viable Listeria species was developed based on reverse transcription-multiplex PCR (RT-MPCR) and restriction digestion. The targets for RT-MPCR were iap mRNAs whose genes are common to all Liseria species. A set of five primers was used in this study. Two of them were genus specific, and the other three were specific to L. monocytogenase, L. innocua, and L. grayi respectively. By RT-MPCR, L. monocytogenese, L. innocua, L. grayi, and a group of Listeria species, including L. ivanovii, L. welshimeri, and L. seeligeri, were specifically identified. To differentiate the latter three Listeria species, RT-MPCR products were subjected to digestion with HpaI and ScaI. The sensitivity of RT-MPCR in detecting Listeria species was determined to be 50 CFU/mL. RT-MPCR was found to discriminate between viable and nonviable cells and to detect viable Listeria species in a food model.

Detection of Aeromonas salmonicida by reverse transcription-multiplex polymerase chain reaction.

Aeromonas salmonicida is one of the major fish pathogens causing economically devastating losses in aquaculture. A. salmonicida subsp. salmonicida is a typical A. salmonicida causing furunculosis, while the other subspecies are atypical strains causing ulcer diseases. PCR-based methods of detecting A. salmonicida suffer from the drawback that they do not distinguish living (pathogenic) from dead cells. In this study, a method of detecting A. salmonicida was developed based on reverse transcription-multiplex PCR (RT-MPCR) using two sets of primers, SV1/SV2 and SF1/SF2, specific to the vapA gene and the fstB gene of A. salmonicida respectively. This method was found to detect A. salmonicida specifically with detection limits of 10 CFU in pure culture and 30 CFU in the presence of tissue debris. It was also found distinguish not only between viable and nonviable cells but also between typical and atypical strains of A. salmonicida. Using RT-MPCR, two DNA fragments, of 542 and 1,258 bp, were amplified from RNA of typical A. salmonicida, whereas only one DNA fragment, of 542 bp, was amplified from the RNA of the atypical ones. The proposed assay was also used successfully to detect A. salmonicida in artificially infected rainbow trout (Oncorhyncus mykiss).

Assessment of factors influencing antimicrobial activity of carvacrol and cymene against Vibrio cholerae in food.

Carvacrol and cymene, phenolic compounds naturally present in the essential oil of oregano and thyme, were examined for their antimicrobial activity against Vibrio cholerae (ATCC 14033, VC1, and VC7) inoculated in carrot juice. Carvacrol exhibited a dose dependent inhibitory effect on the bacteria. Although cymene did not have antimicrobial activity against the bacteria, it enhanced the inhibitory ability of carvacrol. At 25 °C, the lowest concentrations of carvacrol and cymene required for zero detectable viable count varied depending on bacterial strains; 5 and 5 ppm, respectively, for VC7; 5 and 7.5 ppm, respectively, for VC1; and 7.5 and 7.5 ppm, respectively, for ATCC 14033. This study also examined several factors influencing the antimicrobial activity of carvacrol and cymene against V. cholerae ATCC 14033, including temperature, bacterial cell number, and food substrate. Carvacrol and cymene inhibited the bacterium in carrot juice at 25 °C more efficiently than at 15 and 4 °C. The doses of both compounds required for zero detectable viable count increased as the number of the bacterial cells in the carrot juice increased. The fat content and the complexity of foods were shown to decrease the antimicrobial activity of the compounds.

Antimicrobial activity of basil (Ocimum basilicum) oil against Salmonella enteritidis in vitro and in food.

Nine essential oils were examined for antimicrobial activity against reference and clinical strains of Salmonella Enteritidis. Based on the size of the inhibition zone and the minimal inhibitory concentration, basil oil had the strongest antimicrobial activity against all the tested bacteria, and S. Enteritidis SE3 was the most sensitive strain to all the tested oils. Gas chromatography/mass spectrometry analysis revealed that the major constituents of the oil were linalool (64.35%), 1,8-cineole (12.28%), eugenol (3.21%), germacrene D (2.07%), alpha-terpineol (1.64%), and rho-cymene (1.03%). When applied in nham, a fermented pork sausage, experimentally inoculated with S. Enteritidis SE3 and stored at 4 degrees C, basil oil inhibited the bacterium in a dose-dependent fashion. Basil oil at a concentration of 50 ppm reduced the number of bacteria in the food from 5 to 2log cfu/g after storage for 3 d. An unmeasurable level of the bacterium in the food was observed at days 2 and 3 of storage when 100 and 150 ppm of basil oil was used, respectively. Sensory evaluation suggested that the addition of 100 but not of 150 ppm to nham would be acceptable to consumers. The results from this study confirm the potential use of basil oil as an antimicrobial agent to control S. Enteritidis in food.

Synergistic antimicrobial effect of nisin and p-cymene on Salmonella enterica serovar Typhi in vitro and on ready-to-eat food.

Foods contaminated with Salmonella enterica serovar Typhi are a major cause of typhoid fever, leading to public health problems and economic losses worldwide. Nisin and rho-cymene were tested in this study for their antimicrobial activity against S. Typhi at 4 degrees C and 37 degrees C. Nisin and rho-cymene, when used separately, did not inhibit the bacterium at either temperature. A synergistic antimicrobial effect between both compounds was observed when they were used simultaneously. This synergism was greater at 37 degrees C than at 4 degrees C. The lowest concentrations of nisin and rho-cymene required for complete inhibition of S. Typhi at 37 degrees C were 0.3 ppm and 1.5 ppm, respectively, and 0.3 ppm and 2.5 ppm at 4 degrees C. The potential of nisin and rho-cymene to control an S. Typhi population on ready-to-eat Thai-style pork sausage was also examined. The compounds were able to eliminate the contaminating bacterium with concentrations depending on the bacterial cell number on the food.

Potential of coriander (Coriandrum sativum) oil as a natural antimicrobial compound in controlling Campylobacter jejuni in raw meat.

Twelve essential oils were tested in vitro for antimicrobial activities against several strains of Campylobacter jejuni, a pathogen causing food-borne diseases worldwide. Using disk diffusion and minimal inhibitory concentration determination assays, we noted that coriander oil exhibited the strongest antimicrobial activity against all tested strains. The oil had a bactericidal effect on the target bacteria. In evaluating the antimicrobial potency of coriander oil against C. jejuni on beef and chicken meat at 4 degrees C and 32 degrees C, it was found that the oil reduced the bacterial cell load in a dose-dependent manner. The type of meat and temperature did not influence the antimicrobial activity of the oil. This study indicates the potential of coriander oil to serve as a natural antimicrobial compound against C. jejuni in food.

Glass bead transformation method for gram-positive bacteria

A simple, inexpensive and reproducible transformation method was developed for Gram-positive bacteria. It was based on agitation of bacterial protoplasts with glass beads in the presence of DNA and polyethylene glycol. By using this method, introduction of pGK12 into protoplasts of several strains of Gram-positive bacteria was achieved.

Protective effect of clove oil-supplemented fish diets on experimental Lactococcus garvieae infection in tilapia.

The essential oils extracted from the four herbs, cinnamon (Cinnamomum verum), clove (Syzygium aromaticum), ginger (Zingiber officinale) and holy basil (Ocimum sanctum), were investigated for their antimicrobial activity and mode of action against Lactococcus garvieae, a fish pathogenic bacteria causing lactococcosis. Of all the tested oils, clove oil had the strongest inhibitory effect and exhibited a bactericidal mode of action against the pathogenic bacterium. When an intraperitoneal infection of tilapia (Oreochromis niloticus) with L. garvieae was performed, the median lethal dose (LD(50)) was determined to be 1.78x10(2) CFU/fish. For an in vivo trial, no mortality was apparent in fish fed on the fish diets supplemented with 3% (w/w) of clove oil and with 0.5% (w/w) of oxytetracycline 5 d prior to the infection with L. garvieae. These results indicate that clove oil had a protective effect on experimental L. garvieae infection in tilapia and the potential to replace antibiotics for controlling the disease.

Antimicrobial activity of elephant garlic oil against Vibrio cholerae in vitro and in a food model.

Vibrio cholera is a major foodborne pathogen in Thailand. It is present in raw and lightly cooked foods, and it causes cholera. Natural products inhibiting it can be used to improve the safety of foods. In this study, elephant garlic oil was studied for its major diallyl sulfide content and its antimicrobial activity against V. cholerae. The oil had a very low concentration of diallyl monosulfides (1.62%) in comparison with the other diallyl sulfides (25.09% for diallyl disulfide, 16.04% for diallyl trisulfide, and 10.58% for diallyl tetrasulfide). In an in vitro study, the oil was found to have a bacteriocidal effect on all tested strains of V. cholerae, with varied minimal inhibitory concentrations (MICs) ranging from 3.13 to 25 microg/ml. It was also found that elephant garlic oil retarded the growth of the bacteria or reduced the bacterial cell load in the food model, depending on its concentration.

Prophylactic effect of Andrographis paniculata extracts against Streptococcus agalactiae infection in Nile tilapia (Oreochromis niloticus).

Six herbs were assessed for their antimicrobial activity against Streptococcus agalactiae, a major fish pathogen causing streptococcosis. Each herb was extracted with 3 solvents: water, 95% ethanol, and methanol. Using swab paper disc assays, aqueous extracts of Andrographis paniculata and Allium sativum produced the largest (27.5 mm) and smallest (10.3 mm) inhibition zones, respectively. Determination of minimal inhibitory concentration (MIC) of herb extracts against S. agalactiae showed that the aqueous extract of A. paniculata had the lowest MIC value (31.25 microg/mL). Aqueous extract of A. sativum was the only herb extract with a MIC > 500 microg/mL. Based on mortalities in 2 weeks after intraperitoneal S. agalactiae injection, the median lethal dose (LD(50)) of S. agalactiae for Nile tilapia (Oreochromis niloticus) was 3.79 x 10(5) CFU/mL. In vivo experiments showed that fish feed supplemented with either A. paniculata leaf powder or dried matter of A. paniculata aqueous extract reduced mortality of S. agalactiae infected Nile tilapia. In addition, no mortality was found in fish receiving dried matter of A. paniculata aqueous extract supplemented feeds at ratios (w/w) of 4:36 and 5:35. During 2 weeks of feeding with A. paniculata supplemented feeds, no adverse effects on appearance, behavior, or feeding responses were observed.

Glass bead transformation method for gram-positive bacteria.

A simple, inexpensive and reproducible transformation method was developed for Gram-positive bacteria. It was based on agitation of bacterial protoplasts with glass beads in the presence of DNA and polyethylene glycol. By using this method, introduction of pGK12 into protoplasts of several strains of Gram-positive bacteria was achieved.

Potential of Psidium guajava supplemented fish diets in controlling Aeromonas hydrophila infection in tilapia (Oreochromis niloticus).

Fourteen herbs were extracted in water, 95% ethanol, and ether, and tested in vitro for antimicrobial activities against Aeromonas hydrophila, a fish pathogen causing motile Aeromonas septicemia. Using swab paper disc assays and minimal inhibitory concentration (MIC) determinations, we noted that the ethanol extract of Psidium guajava leaf exhibited the strongest antimicrobial activity. The extract allowed growth-inhibited A. hydrophila cells to regrow in fresh BHI broth indicating a bacteriostatic mode of action. In a pathogenicity test, the median lethal dose (LD(50)) of A. hydrophila for tilapia (Oreochromis niloticus) by intraperitoneal injection was 3.44 x 10(6) CFU/ml. In vivo experiments showed that fish diets containing either dry leaf powder of P. guajava or dried ethanol extract of P. guajava leaf reduced mortality of A. hydrophila infected tilapia with no detected adverse effect on the fish. This study suggests that P. guajava leaf has the potential to control fish diseases caused by A. hydrophila.

Diallyl sulfide content and antimicrobial activity against food-borne pathogenic bacteria of chives (Allium schoenoprasum).

Chives, a member of the Alliaceae family, have been used in food and medicine in Thailand for a long time. Diallyl sulfides (diallyl monosulfide, dially disulfide, diallyl trisulfide, and diallyl tetrasulfide) are believed to be responsible for the antimicrobial activity of plants in this family. In this study, chive oil was examined for its diallyl sulfide content and its antimicrobial activity against some strains of food-borne pathogenic bacteria. Chive oil had a very low concentration of diallyl monosulfide in comparison with the other diallyl sulfides. They inhibited all pathogenic bacteria used in this study with a different degree of inhibition. Chive oil was also shown to be able to inhibit Escherichia coli O157:H7 in a food model. This study is the first report describing not only the diallyl disulfide content of chive oil, but also its antimicrobial activity against food-borne pathogens in both a test tube and food model.

Incidence of nisin Z production in Lactococcus lactis subsp. lactis TFF 221 isolated from Thai fermented foods.

Lactic acid bacteria isolated from various Thai fermented foods were screened for the presence of nisin gene by using PCR with primers specific to nisin A structural gene. Only one strain, Lactococcus lactis subsp. lactis TFF 221, isolated from kung jom, a traditional shrimp paste, was found to carry a nisin gene. The TFF 221 nisin had antimicrobial activity against not only closely related lactic acid bacteria but also some foodborne pathogens. It was heat stable and inactivated by alpha-chymotrypsin and proteinase K. Some characteristics of TFF 221 nisin were found to be very similar to those of nisin A produced by Lactococcus lactis subsp. lactis NCDO 2111. Both of them had the same antimicrobial spectrum and MICs against all indicator bacteria. However, when assayed with indicator organisms, in all cases the TFF 221 nisin produced larger zones of inhibition in agar diffusion assays than the nisin A did. Sequencing of the TFF 221 nisin gene showed that it was the natural nisin variant, nisin Z, as indicated by the substitution of asparagine residue instead of histidine at position 27. The nisin determinant in strain TFF 221 was found to be located on a conjugative transposon residing in the chromosome. The ability of the nisin produced by L. lactis subsp. lactis TFF 221 to inhibit a wide range of foodborne pathogens may be useful in improving the food safety of the fermented product, especially in the Thai environment, which suffers from perennial problems of poor food hygiene.

Bacteriostatic effect of flavonoids isolated from leaves of Psidium guajava on fish pathogens.

The antimicrobial activity against fish bacterial pathogens of flavonoids (morin, morin-3-O-lyxoside, morin-3-O-arabinoside, quercetin, and quercetin-3-O-arabinoside) isolated from the leaves of Psidium guajava was evaluated. The flavonoids were shown to have bacteriostatic effect on all of the tested bacteria.