Growth Conditions Affecting Proteolytic Enzyme and Extracellular Vesicle Production by Pseudomonas fragi ATCC 4973

Pseudomonas fragi ATCC 4973 was grown in liquid culture (trypticase soy broth) and on a solid surface (tryticase soy broth + 1.5% agar). Pseudomonas fragi grown in liquid culture started extracellular proteolytic enzyme production at 24 hr, during the late exponential early stationary growth phase. Pseudomonas fragi, grown on solid surfaces, initiated proteinase production at 4 hr, 20 hr earlier than in liquid medium. Proteinase production was greatest during periods when the cells were not actively growing. Electron micrographs of P. fragi grown on solid surfaces revealed extracellular vesicles ca. 20 nm in diameter “blebbing” off the surface of the cells. The greatest number of vesicles coincided with maximum proteinase production. These vesicles were absent from the surface of P. fragi cells grown in liquid culture.     

Electron Microscopic Investigation of Pseudomonas fragi ATCC 4973 on Intact and Sarcoplasm-Depleted Bovine Longissimus dorsi Muscle at 21°C

Scanning and transmission electron microscopy were used to investigate formation and role of glycocalyx material involved in adhesion of Pseudomonas fragi to intact and sarcoplasm-depleted beef surfaces. Depletion of sarcoplasm did not decrease attachment of P. fragi to bovine muscle. P. fragi caused a rapid increase in pH of only intact muscle. Examination of inoculated muscle (washed and intact) by SEM after 1, 2, 3, or 5 day incubation period, revealed a pebbling effect on the bacterium surface as well as a coiling of glycocalyx material. TEM showed two types of polymeric material; one was adherent to the bacterial surface while the other was amorphous. The amorphous type probably corresponded to the coiled glycocalyx revealed by SEM. Close association between glycocalyx and bleb-like evaginations on P fragi reinforces previous hypotheses concerning their role and functions in attachment and meat spoilage.     

Colonization of Beef Muscle Surfaces by Pseudomonas fluorescens and Pseudomonas fragi

Attached and unattached cell densities were determined for Pseudomonas fluorescens and Pseudomonas fragi growing on the surface of beef muscle stored at 4 and 25°C, in presence of NaCl, KCl, and CaCl₂. A mechanical rinsing procedure was developed for this purpose. Both species colonized the surface at both temperatures and were enhanced at low (4°C) temperature. Attached cells represented up to 90% of the total until a density of 10⁵-10⁶ CFU cm^?2 was reached. At that point, a proportion of attached cells to unattached cells declined but colonization of the surface continued. In presence of CaCl₂, ratios of attached to unattached cells did not decline, suggesting a significant role for the calcium ion in colonization. Ability to colonize meat surfaces may be a significant competitive advantage for meat spoilage bacteria such as Pseudomonas fragi and Pseudomonas fluorescens.     

Some Biochemical Changes in Sarcoplasmic Depleted, Intact Beef Muscle Inoculated with Pseudomonas frari

Twenty-four hour postmortm intact bovine Longissimus dorsi muscle strips were (1) washed to remove sarcoplasmic fluid, or (2) left intact, and were either uninoculated or inoculated with Pseudomonas fragi ATCC 4973 to evaluate the effect of decreased concentration of sarcoplasm on bacterial growth and subsequent spoilage during storage at 4°C for 12 days. Washing removed the majority of the water-soluble components of the muscle. Significantly (P < 0.01) higher growth rates were observed on intact muscle than on washed muscle. Increased extractability of water-soluble and salt-soluble proteins was observed in intact inoculated (II) muscle as growth of P. fragi progressed. Alterations in SDS-gel electrophoretic patterns of water-soluble, salt-soluble, urea-soluble and urea-insoluble proteins were evident in the II muscle. Relatively little change in nonprotein nitrogen, water-soluble and salt-soluble protein content occurred in washed inoculated (WI) muscle with increased bacterial growth. Only minor changes in the SDS-gel electrophoretograms of the salt-soluble proteins from the WI tissue were apparent.     

Proteolytic activity of four strains of Pseudomonas on crude extracts of contractile proteins

Hydrolytic activities of exogenous proteases obtained from Pseudomonas upon crude extracts of contractile proteins were studied. Four strains were used: C61 and C20, isolated from beef samples, and P. fragi (ATCC-4973) and P. fluorescens (NRRL-B-1244). Proteolytic activity was tested on crude extracts of actomyosin, myosin+ actin+regulatory proteins, actin and high molecular weight proteins obtained from postrigor beef by differential precipitation according to their solubility in salt solutions of various ion strengths. Electrophoresis analysis showed considerable degradation of actomyosin in samples treated with a C61 enzymatic extract. Myosin degradation was similar for treatments with proteases obtained from C61 and P. fragi. Actin degradation was similar for all enzymatic extracts. The most active protease was a 46.8 kDa enzyme produced by C60. P. fragi produced two proteases of 49.2 kDa and 34.2 kDa, P. fluorescens one of 46.1 kDa, and C20 one of 49.2 kDa.     

Productions and monomer compositions of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains isolated from traditional home-made yoghurts and raw milk

In this study, a total of forty‐five strains of lactobacilli and streptococci were determined exopolysaccharide (EPS) production in skim milk and Man Rogosa and Sharpe (MRS)/M17 medium, viscosity and proteolytic activity. The exopolysaccharide production by lactobacilli strains during growth in MRS medium was twenty‐one to 211 mg L^?1, while in skim milk was to thirty‐six to 315 mg L^?1. The EPS production by streptococci strains during growth in M17 medium was sixteen to 114 mg L^?1, while in skim milk was to twenty‐four to 140 mg L^?1. The EPS production of strains was lower in MRS/M17 medium than skim milk. Results showed that it was not clear correlation between the viscosity and EPS production of some strains. All strains were shown proteolytic activity. Positive correlations between exopolysaccharide production and proteolytic activity in skim milk were found some strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These results indicated that the high exocellular protease‐producing strains can produce high EPS in skim milk. The monomer compositions of the EPSs formed by selected five strains were analysed. Mannose dominated (99–100%) on the EPS produced by L. delbrueckii subsp. bulgaricus and S. thermophilusstrains (except L. delbrueckii subsp. bulgaricus 22) in skim milk and MRS/M17 medium. Besides, the EPSs of strains in skim milk contained small amount of lactose.     

Predictive Microbiology for Monitoring Spoilage of Dairy Products with Time-Temperature Integrators

Time/temperature integrators (TTI) have a potential for monitoring time-temperature history of perishable foods, including dairy products. To correlate the end of shelf life of dairy products with different TTI's, kinetic data for growth of a dairy spoilage microorganism was obtained. Both Arrhenius and square root equations were used to model the growth of Pseudomonas fragi. A significant negative history effect was observed for P. fragi growth rate whereas history effect was positive on the lag phase, under certain nonisothermal conditions. A correlation scheme of P. fragi growth was developed with the TTI response. The application of TTI's for dairy products is feasible despite the history effects.     

Effect of Various Agents Upon the Attachment of Pseudomonas fragi to Stainless Steel

Epifluorescent microscopy was used to assay for the effect of selected chemicals and enzymes upon physical attachment of Pseudomonas fragi to stainless steel. The assays were conducted after the pretreatment of cells with the agents, in the presence of agents, and in the removal of attached cells with test agents. Compared to the controls, sodium periodate, cetyltrimethylammonium bromide (Cetavlon), NaOH, and Concanavalin A inhibited the attachment process in the pretreatment and presence assays. Trypsin, sodium dodecyl sulfate (SDS), NaOH and sodium periodate were effective in removing attached cells, while Cetavlon and Triton X-100 stabilized the attachment of cells to the stainless steel surface and prevented their removal. Neither EDTA nor mechanical removal of flagella had a significant effect upon attachment. These results suggested that attachment of P. fragi to stainless steel involved a polysaccharide and protein matrix surrounding the cells.     

Pseudomonas spp. convert metmyoglobin into deoxymyoglobin

Meat ‘reddening’ by bacteria was observed in chilled beef. To identify the reddening bacteria, isolates were inoculated onto beef and the changes in CIE L^*a^*b^* values monitored. As a result, two Pseudomonas spp., including Pseudomonas fragi which is commonly observed in raw meat, were selected and identified as reddening bacteria. The reddening was coincidentally occurred with the appearance of slime, and the increase in thiobarbituric acid-reactive substances (TBARS) was simultaneously suppressed. In myoglobin-containing nutrient broth, it is shown spectroscopically that P. fragi converted metmyoglobin into deoxymyoglobin. It was concluded that the meat reddening was due to the formation of deoxymyoglobin, induced by the very-low-oxygen tension brought about by Pseudomonad’s oxygen consumption: This oxygen depletion simultaneously suppressed TBARS increase.     

The microflora of fresh and spoiled sardines (Sardina pilchardus) caught in Adriatic (Mediterranean) Sea and stored in ice

A total of 1500 strains were isolated from the skin and gills of fresh, ice-stored (4 days) and spoiled (8 days) Adriatic sardines, and were identified at different taxonomic levels. Fresh sardine microflora found included mostly non-fermenting Gram-negative bacteria, (Pseudomonas, Flavobacterium, Psychrobacter, Acineobacter, Shewanella), and a minor proportion of Enterobacteriaceae and Gram-positive bacteria. The highest increase in microflora frequency, after 8 days in ice, was observed for thePseudomonas fragigroup (8–30·8%) andShewanella putrefaciens(1·8–11%). A significant presence was also noted for fluorescentPseudomonas(15·6–18·4%) andPsychrobacter immobilis(16·4–23·4%). The isolation frequency of the other groups decreased during storage. The most important proteolytic species werePseudomonas fluorescensandShewanella putrefaciensand the most lipolytic bacteria werePsychrobacter immobilisand again,P. fluorescensandS. putrefaciens. A total of 288 isolates, representative of the main groups, were tested for potential spoiling activity (H₂S and off-odour production, TMAO reduction).Shewanella putrefacienswas the strongest spoiler, followed byPseudomonas fluorescens. A weaker activity was observed for strains ofPseudomonas fragi, enterobacteriaceae and non-saccharolytic pseudomonads. The contribution of weak spoiler bacteria can be undermined by the activity of the strongest spoilers, but in some cases the considerable incidence of the former group suggests their effective role.Morganella morganiiwas the only histamine-producing species among 57 screened strains representative of different taxa.     

Exopolysaccharides produced by Lactobacillus sp.: Biosynthesis and applications

Lactobacillus sp. synthesize exopolysaccharides (EPS), including both homo- and heteropolysaccharides, which play an important role in the production of fermented foods, and especially in the dairy industry, improving the gustatory and rheological properties of the finished products. These polymers are generated by starter cultures in situ in fermented foods, and so they are treated as natural thickening agents. As some Lactobacillus strains are generally recognized as safe and have been shown to exhibit probiotic activity, EPS from those bacteria can be used as functional food ingredients, conferring both health and economic benefits to the consumers. However, their industrial applications are hindered by the low yield of EPS from Lactobacillus and high costs of their purification. This review focuses on the latest reports concerning the biosynthesis and properties of Lactobacillus EPS.     

Effect of the extract from custard apple (Annona squamosa) leaves prepared with pulsed electric field-assisted process on the diversity of microorganisms and shelf-life of refrigerated squid rings

Antibacterial and antibiofilm properties of ethanolic custard apple leaf extract (CALE) prepared without and with pulsed electric field (PEF) assisted process (6 kV cm⁻¹, 300 pulses, 142 kJ kg⁻¹ for 5 min) (PEF-CALE) were comparatively investigated. PEF-CALE showed higher bacterial inhibition against Bacillus subtilis, Listeria monocytogenes, Escherichia coli, and Pseudomonas aeruginosa, compared to CALE. Antibiofilm properties of both extracts towards the aforementioned bacteria were augmented with increasing concentrations (P < 0.05). The impact of PEF-CALE in extending the shelf-life of squid rings (SQR) was also investigated. Based on the microbiological limit, the shelf-life of refrigerated squid rings pretreated with 400 mg kg⁻¹ PEF-CALE was extended by more than 6 days when compared to the control. Pseudomonas lundensis, Pseudomonas fragi, and Shewanella baltica were the most abundant spoilage bacteria found in SQR. Overall, PEF-CALE exhibited promising antimicrobial activities, which could serve as biopreservative agent for inhibiting bacterial growth in SQR during refrigerated storage.     

The effect of modified atmosphere packaging on the microbiological,physical, chemical and sensory characteristics of broadtail squid (Illex coindetii)

The aim of this work was to evaluate the effect of various atmosphere compositions (20% CO₂/80% N₂ for modified atmosphere packaging (MAP) 1, 50% CO₂/50% N₂ for MAP 2, 70% CO₂/30% N₂ for MAP 3 and vacuum packaging) on the microbial (mesophiles, psychrophiles, Pseudomonas spp., Brochothrix thermosphacta and Enterobacteriaceae), physical, chemical [trimethylamine (TMA) and total volatile basic nitrogen (TVBN)] and sensorial characteristics of broadtail squid (Illex coindetii) stored for 10 days at 2 ± 1 °C. All microbial populations were severely restrained by MAP 3 with the exception of Enterobacteriaceae, which seemed to take advantage of the lack of competitive microflora and had enhanced microbial counts on MAP samples (P < 0.05). Colour attributes were better maintained on MAP‐stored samples. Drip loss was less on vacuum‐packaged squids. MAP 2 was the best atmosphere for the preservation of tissue consistency. TMA and TVBN formation was limited by high CO₂ atmospheres, even though both elevated in all studied conditions. Shelf life based on sensory characteristics was determined to be 10, 8, 6, 6 and 4 days for MAP 3, MAP 2, MAP 1, vacuum and control samples, respectively.     

Isolation and identification of exopolysaccharide producer lactic acid bacteria from Turkish yogurt

Lactic acid bacteria (LAB) were isolated from traditional yogurt samples and genotypic characterization of these isolates revealed the presence of 21 distinct LAB strains belonging to Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Leuconostoc mesenteroides, and Lactobacillus plantarum as new LAB strains. Determination of the exopolysaccharide (EPS) production characteristics of the selected strains of each species revealed that all strains possessed at least one gene required for both homopolymeric‐ and heteropolymeric‐type EPS production. Structural analysis of the EPSs showed that L. delbrueckii subsp. bulgaricus Y39 and S. thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuc. mesenteroides Y35 and L. plantarum Y36 produced homopolymeric glucan‐type EPS. The level of EPS production in these strains was found to be in a similar range. These strains with EPS production characteristics are good candidates for future studies as new LAB for yogurt production.

Practical applications

Recent trends in yogurt production technology have led to an increased use of ropy starter cultures in yogurt production due to the technological roles of exopolysacharides (EPS) produced by these cultures. The main role of EPS in yogurt production is to improve the textural properties of yogurt as an in situ produced natural polymer. In addition to the yogurt starter cultures, use of adjunct cultures during production of yogurt is also of special interest to enhance the technological and nutritional characteristics of yogurt. Therefore, in this study, potential yogurt starter and adjunct cultures from traditional yogurt samples with EPS production characteristics were isolated. From these isolates, Lactobacillus delbrueckii subsp. bulgaricus Y39 and Streptococcus thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuconostoc mesenteroides Y35 and Lactobacillus plantarum Y36 produced homopolymeric glucan.     

Evaluation of the yield,molar mass of exopolysaccharides,and rheological properties of gels formed during fermentation of milk by Streptococcus thermophilus strains St-143 and ST-10255y

The yield and chemical structures of exopolysaccharides (EPS) produced by many strains of Streptococcus thermophilus have been characterized. However, the kinetics (or production profile) for EPS during milk fermentation is not clear. In this study, we investigated whether any differences existed in the yield and molar mass of EPS when milk was fermented at the same acidification rate by 2 strains of S. thermophilus (St-143 and ST-10255y). The type of EPS produced by these 2 strains is different. Milk samples were analyzed for EPS concentration every 30 min during a fermentation period of 270 min (final pH 4.5) by using a modified quantification method, which was faster and validated for its recovery of added EPS. Rheological properties of milks during fermentation were also analyzed using small-strain dynamic oscillatory rheology. For the determination of molar mass, EPS extracts were isolated by ultrafiltration of whey obtained during fermentation of milk to pH values 5.2, 4.9, 4.7, and 4.5, and molar mass was analyzed using size-exclusion chromatography–multi-angle laser light scattering. During fermentation, both strains appeared to start producing significant amounts of EPS after about ～150 min, which corresponded to pH ～5.3, which was close to the point of gelation. During the remainder of the fermentation process (150–270 min), the EPS concentration from strains St-143 and ST-10255y significantly increased from 30 to 72 mg/L and from 26 to 56 mg/L, respectively. The quantity of EPS recovered by our modified method was estimated to represent ～60% of the total EPS added to milk. The molar mass of EPS produced by both strains appeared to slightly decrease during fermentation. At pH 5.2, EPS from St-143 and ST-10255y had molar masses of 2.9 × 10⁶ and 1.4 × 10⁶ g/mol, respectively, which decreased to 1.6 × 10⁶ and 0.8 × 10⁶ g/mol, respectively, when the pH of milk was 4.5. Distinct differences were apparent in the rheological properties of gels fermented by the 2 strains. At the end of fermentation, St-143 fermented milk had weaker gels with storage modulus (G′) value at pH 4.6 of 26 Pa, whereas gels made with ST-10255y were stiffer with a G′ value at pH 4.6 of 82 Pa. For St-143 gels, maximum loss tangent (LT_max) values were higher (0.50) and occurred earlier (at a higher pH value) than the LT_max values (0.46) for gels from ST-10255y strain. Because the fermentation conditions were identical for both strains, the observed changes in rheological properties could be due to the differences in chemical structures and molar mass of the EPS produced by these 2 S. thermophilus strains.     

Adhesion and retention of a bacterial phytopathogen Erwinia chrysanthemi in biofilm-coated porous media

The goal of this study is to investigate the impact of biofilm physical and biological properties on bacterial transport and deposition in porous media. Experiments were performed in packed columns to examine the removal of Erwinia chrysanthemi (Ech3937), a phytopathogen, from the bulk fluid due to its attachmentto glass beads coated with Pseudomonas aeruginosa biofilms. Two isogenic P. aeruginosa strains, PAO1 and PD0300, with different EPS secretion capabilities and EPS compositions, were used to culture biofilms. The Ech3937 transport and distribution in packed columns were studied in both upflow and downflow cell injection modes over a range of solution ionic strengths. The results show that the presence of biofilm strongly interferes with the deposition behavior of Ech3937 in porous media. The spatial variation of deposited Ech3937 cells contradicts the log-linear pattern predicted by the classic filtration theory, indicating that the biofilm physical structure and polymeric interactions between the biofilm EPS and Ech3937 cell surface are the main mechanisms that control bacterial deposition. When the biofilm accumulation is relatively small, bacterial adhesion onto biofilm-coated porous media is mainly inhibited by steric forces. By contrast, cell deposition is enhanced by the reduced porous media porosity when biofilm is more abundant.     

Effect of Cleaning and Sanitizing on the Attachment of Pseudomonas fragi to Stainless Steel

The effects of two clean-in-place (CIP) procedures on attachment of Pseudomonas fragi ATCC 4913 to a stainless steel surface were studied using scanning electron microscopy (SEM). Under normal CIP conditions those cells that remained in the system did not exhibit attachment fibrils. Under conditions of lowered detergent water temperature and detergent and sanitizer concentrations, those cells that were present after CIP showed attachment fibrils. Organisms remaining after normal CIP were not viable, whereas those present after suboptimum cleaning were. These viable cells produced attachment fibrils within 24 hr at 21°C and 96 hr at 4°C when grown in milk.     

Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.     

Effects of Different Gas Compositions on the Color Estimations of MAP Packaged Pork Chops

This study was conducted to observe effects of different gas compositions on color of pork chops packaged in modified atmospheres. Gaseous compositions used were: MAP1 (75% O₂:25% CO₂); MAP2 (70% O₂:30%CO₂) and MAP3 (80% O₂:20% CO₂). Sensory evaluations of meat color and chemical properties (acid number, peroxide value, a_w, pH, TVB-N and TBARs), were carried on the 1^st, 5^th, 7^th, 9^th and 12^th day of storage. The sensory evaluations of chop color in different MAP compositions are analyzed in relation to storage period, measured chemical properties and instrumental determination of meat color using linear and multivariate linear regression analysis.