Contribution to the knowledge about the presence and role of Enterobacter gergoviae in sensory characteristics of dairy products

Enterobacter gergoviae KGPMF 20 was found in traditionally made cheese from Sokobanja (South-eastern Serbia). In this paper, the characteristics of the species were evaluated by investigation of adhesion to different solvents and co-aggregation ability with other species. Moreover, its enzymatic activity was evaluated by using spectrophotometric method, with the intention to detect the role of the isolate in the sensory characteristic of cheese. The results of enzymatic activity indicated that E. gergoviae KGPMF 20 has low, almost no enzymatic activity. It could be concluded that this isolate did not affect the sensory characteristic of cheese.Publishe

Fiziološka karakterizacija bakterija mlečne kiseline izolovanih iz autohtonog sira jugoistočne Srbije i evaluacija njihovih biotičkih potencijala

U ovom rukopisu, ispitano je i utvrđeno prisustvo i fiziološke karakteristike bakterija mlečne kiseline (BMK) izolovanih iz tradicionalno proizvedenog sira iz jugoistočne Srbije (područje Sokobanje). Tri uzorka, iz različitih seoskih domaćinstava, uzorkovana su tokom proleća, leta i jeseni. Ispitane su hemijske karakteristike uzoraka sira. Identifikacija BMK je izvršena korišćenjem biohemijskih testova i MALDI-TOF masene spektrofotometrije. Utvrđen je antimikrobni potencijal u odnosu na rast Escherichia coli ATCC 25922 i Proteus mirabilis ATCC 12453, kao i na rast nekih Gram-negativnih bakterija, izolovanih iz istog sira (Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 i Aeromonas hydrophila KGPMF64), korišćenjem metode difuzije iz bunarića u agar. Za dalje istraživanje, odabrano je 16 izolata, na osnovu prethodno istraženih i utvrđenih biohemijskih karakteristika i antimikrobnog potencijala u odnosu na izabrane enterobakterije. Izvršena je procena probiotskog potencijala odabranih BMK (tolerancija na različite gastrointestinalne uslove i osetljivost na klinički relevantne antimikrobne agense). Ispitan je uticaj različitih vrsta šećera na rast izolovanih BMK, sposobnost formiranja biofilma, sposobnost bakterija da se vezuju za rastvarače i epitelne ćelije tankog creva svinje, kao i sposobnost auto- i koagregacije između njih i kliničkog izolata Escherichia coli. Aktivnost enzima (kisela i alkalna invertaza, alkalna fosfataza, alkalna proteaza, invertaza) kao i ukupna koncentracija proteina u fermentacionoj tečnosti je izmerena spektrofotometrijskom metodom. Procenjen je uticaj različitih temperatura u sinergizmu sa različitim pH i koncentracijama soli na planktonski rast izolata BMK. Prema procentu masnoće u suvoj materiji, sokobanjski sir pripada punomasnoj grupi sireva, dok prema sadržaju vlage u materiji bez masnoće, sokobanjski sir pripada kiseloj grupi sireva. Brojnost bakterija je zavisila od sezone, domaćinstva i vrste podloge koja se koristila za izolaciju i brojanje. Identifikovana su četiri različita roda BMK (Lactobacillus, Lactococcus, Enterococcus i Streptococcus). U okviru roda Lactobacillus identifikovani su Lb. fermentum, Lb. brevis i Lb. plantarum. U okviru roda Lactococcus identifikovanini su L. lactis subsp. lactis i L. lactis subsp. lactis biovar. diacetylactis. U okviru roda Enterococcus identifikovani su E. faecium, E. faecalis, E.II hirae i E. durans. U okviru roda Streptococcus identifikovani su S. uberis i S. agalactiae. Svi testirani izolati, osim izolata iz roda Streptococcus, pokazali su dobru acidifkacionu sposobnost u standardnom i obogaćenom mleku. Testirani izolati su inhibirali rast bar jedne od pet indikatorskih vrsta, sa prečnikom zone inhibicije od 10 do 26 mm. S. uberis (7 izolata) i S. agalactiae (1 izolat) su izolovani iz sira uzorkovanog tokom leta. Testirani Streptococcus izolati su pokazali osetljivost na tetraciklin, hloramfenikol, novobiocin i rifampicin, sa prečnikom zone inhibicije od 36 do 48 mm. Zbog specifičnosti tradicinalnog načina proizvodnje sokobanjskog sira, smatra se da su ovi izolati dospeli u mleko, a kasnije u sir sa vimena krava. Izabrani izolati BMK su pokazali toleranciju na simulirane gastrointestinalne uslove i osetljivost na antibiotike, posebno na ampicilin (MIK na 0,195 μg mL-1 za laktobacile; od 0,195 – 3,125 μg mL-1 za laktokoke; od 0,19 – 2,5 μg mL-1 za enterokoke). Sposobnost formiranja biofilma izolata BMK je bila izolat specifična. Najveći procenat adhezije je detektovan sa hloroformom, dok je adheziona sposobnost odabranih izolata na epitel tankog creva svinje u korelaciji sa rezultatima adhezione sposobnosti prema rastvaračima. Sposobnost autoagregacije izolata je bila jaka. Lactobacillus spp. su pokazali snažnu koagregaciju sa E. coli, dok je koagregacija izolata iz rodova Lactococcus i Enterococcus bila izolat specifična. Svi izolati iz roda Lactobacillus su pokazali aktivnost proteaze, amilaze i alkalne fosfataze, dok aktivnost kiselih i alkalnih invertaza nije uočena. Izolati iz roda Lactococcus su pokazali aktivnost proteaze, kisele invertaze i alkalne fosfataze, osim izolata KGPMF50, koji nije pokazao aktivnost alkalne fosfataze. Izolati iz roda Enterococcus su pokazali slabu i izolat specifičnu enzimsku aktivnost. Proteini su detektovani u fermentacionoj tečnosti svih izolata. Izolati iz rodova Lactobacillus i Lactococcus su pokazali veću toleranciju na kiselu pH, dok su izolati iz roda Enterococcus bili tolerantniji na baznu pH, na 37°S. Temperatura na 4°S je bila limitirajući faktor za rast svih testiranih izolata. Ograničavajući faktori za planktonski rast izolata iz rodova Lactobacillus i Lactococcus su bili bazna pH, koncentracija soli iznad 6,5% i temperatura na 20°S. Ograničavajući faktori za rast izolata iz roda Enterococcus su bili kisela pH, koncentracija soli oko 8% i temperatura na 20°S. U kombinaciji sa 8% soli i temperaturom od 20°S, samo su izolati iz roda Lactococcus pokazali minimalanu sposobnost rasta, dok izolati iz roda Lactobacillus nisu pokazali sposobnost rasta.III Rezultati su ukazali na potencijalna probiotska svojstva BMK izolovanih iz sokobanjskog sira i otvorila mogućnost za dalja istraživanja i potencijalnu primenu u mlečnoj industriji. Izolati su uglavnom pokazali visoku toleranciju na ekstremne uslove koji vladaju u gastrointestinalnom traktu, osetljivost na antibiotike, kao i sposobnost preživljavanja u prisustvu fenola. Izolati nisu pokazali sposobnost da sintetišu histamin i tiramin, što je poželjna karakteristika pri izboru mogućih probiotika ili starter kultura. Na osnovu rezultata sposobnosti adhezije i agregacije, izdvojili su se izolati Lb. fermentum KGPMF28, Lb. fermentum KGPMF29, Lb. brevis KGPMF35 i L. lactis subsp. lactis biovar. diacetylactic KGPMF57, koji su pokazali potencijal za upotrebu i buduća istraživanja.In this paper, the presence and physiological characteristics of lactic acid bacteria (LAB) isolated from traditionally made cheese from Southeastern Serbia (Sokobanja area) were examined. Three samples, from different households, were obtained during the spring, summer and autumn. Chemical characteristics of cheese samples were examined. Identification of LABs was done by using the biochemical tests and MALDI-TOF mass spectrometry. The antimicrobial potential on the growth of Escherichia coli ATCC 25922 and Proteus mirabilis ATCC12453 as well as on the growth of some Gram‐negative bacteria, isolated from the same cheese (Klebsiella oxytoca KGPMF1, Klebsiella ornithinolytica KGPMF8 and Aeromonas hydrophila KGPMF64) was examined by using agar-well diffusion method. The 16 isolates were chosen for further investigation, according to the previously investigated biochemical characteristics and antagonistic potential against enterobacteria isolated from the same cheese. The evaluation of the probiotic potential of chosen LABs (tolerance to different gastrointestinal conditions and sensitivity to clinically relevant antimicrobial agents) was evaluated. The effect of different type of sugars on the growth of isolated LAB, was examined. The ability of biofilm formation of LAB, the ability of bacteria to adhere to solvents and to pig intestinal epithelium, as well as the ability of auto- and co-aggregation between them and Escherichia coli clinical isolate, were investigated. The enzymatic activities (acid and alkaline invertase, alkaline phosphatase, alkaline protease, invertase) as well as the total concentration of protein in fermentation liquid were measured by using the spectrophotometry method. The effect of different temperature in synergism with different pH and salt concentration, on the ability of planktonic growth of selected LAB, was also evaluated. According to the fat in dry matter, the cheese belong to full-fat, while according to moisture in nonfat substance in acid-curd soft cheese group. The number of viable bacterial cells showed seasonal, household and type of substrate dependence. Briefly, four different genera of LAB (Lactobacillus, Lactococcus, Enterococcus and Streptococcus) were identified. The members of the genus Lactobacillus were Lb. fermentum, Lb. brevis and Lb. plantarum. The members of the genus Lactococcus were L. lactis subsp. lactis and L. lactis subsp. lactis biovar. diacetylactis. The members of the genus Enterococcus were E. faecium, E. faecalis, E. hirae and E. durans. The members of genus Streptococcus were S. uberis and S. agalactiae. All tested isolates, except the members of genus Streptococcus, showed good acidification ability in pure and enriched milk. Tested isolates inhibited the growth of at least one indicator strain. Diameter of the growth inhibition zone was from 10 to 26 mm.V d S. agalactiae (1 isolate) were isolated from the cheese samples taken in summer. All isolates were susceptible to tetracycline, hloramphenicol, novobiocin and rifampicin, with a growth inhibition zone from 36 – 48 mm. Due to the specific making of cheese from Sokobanja, these isolates are, probably, originating from cows’ udders. Chosen isolates were tolerant to the simulated gastrointestinal condition and showed sensitivity to antibiotics, especially to ampicillin (MIC at 0.195 μg mL-1 for lactobacilli; from 0.195 – 3.125 μg mL-1 for lactococci; from 0.19 – 2.5 μg mL-1 for enterococci). The sugar metabolism and the ability of biofilm formation of LABs were strain specific. The highest percentage of adhesion was detected with chloroform, while the adhesion ability of selected isolates to pig intestinal epithelium was in the correlation with the results of adhesion ability to solvents. The auto-aggregation ability of isolates was strong. Lactobacillus spp. demonstrated hight co-aggregation with E. coli, while co-aggregation of Lactococcus spp. and Enterococcus spp. was strain specific. All Lactobacillus isolates showed the protease, amylase and alkaline phosphatase activity, while the activity of acid and alkaline invertase was not observed. The Lactococcus isolates showed the protease, acid invertase and alkaline phosphatase activity, except KGPMF 50 isolate, which showed no alkaline phosphatase activity. Tested Enterococcus isolates showed weak and strain specific enzymatic activity. The proteins were detected in the fermentation liquid of all isolates. Tested Lactobacillus and Lactococcus isolates showed tolerance to acidic pH, while Enterococcus isolates showed tolerance to basic pH, at 37°C. Temperature at 4°C was limiting factor for the growth of all tested bacteria. The limiting factors for the planktonic growth of Lactobacillus and Lactococcus isolates were basic pH, salt concentration above 6.5% and temperature at 20°C. The limiting factors for the growth of Enterococcus isolates were acidic pH, salt concentration around 8% and temperature at 20°C. In combination of 8% of salt and 20°C, only Lactococcus isolates showed minimal growth, while Lactobacillus isolates showed no growth at all. These results indicated the potential probiotic properties of the LABs isolated from Sokobanja cheese and give evidence for further potential application in the dairy industry. Isolates mostly showed a high tolerance to extremes of gastrointestinal conditions, sensitivity to antibiotics, as well as the ability of surviving in the presence of phenol. They showed no ability to synthesize histamine and tyramine, which is desirable characteristic when selecting possible probiotics or starter cultures. Based on the results of adhesion and aggregation ability, Lb. fermentum KGPMF28, Lb. fermentum KGPMF29, Lb. brevis KGPMF35 and L. lactis subsp. lactis biovar. diacetylactic KGPMF57 isolates showed the potential for usage and future investigation

Tolerance of autochthonous lactic acid bacteria to different processing conditions in vitro

In this paper, the effect of different temperatures, pH, and NaCl concentration on the growth of autochthonous lactic acid bacteria isolated from traditionally made Serbian cheese (Sokobanja area) was investigated by using the spectrophotometric method. Growth of tested Lactobacillus (Lb. fermentum, Lb. plantarum, and Lb. brevis) and Lactococcus lactis subsp. lactis biovar. diacetylactis five isolates were better in acidic pH, while the growth of Enterococcus isolates (E. durans, E. faecium, and E. faecalis) was better in basic pH, at 37 °C. At 4 °C after 24 h, none of the tested bacteria showed growth. Since the autochthonous isolates were tolerant to a tested range of dairy processing conditions, further studies need to include the characterization of enzymatic activity of selected isolates, as well as the ability to use these isolates like starter cultures or food supplements in dairy or non-dairy products.Accepted for publishin

The ability to use sugars and sugar substitutes as prebiotics by Serbian autochthonous lactic acid bacteria

The purpose of this study was the investigation of the growth ability of lactic acid bacteria (LAB), isolated from traditionally made cheese from Southeastern Serbia, in the presence of different percent of sugars and sugar substitutes (fructose, lactose, inulin, and mannitol). Dextrose was included for comparative purposes. The results indicated that fructose and lactose stimulated the growth of Lactobacillus strains, with exceptions of Lactobacillus plantarum KGPMF62 and Lb. plantarum LP 299v. Lactose showed similar effects like dextrose on the growth of Lactococcus strains, while higher growth rates of Enterococcus hirae KGPMF9, E. durans KGPMF10, E. faecium KGPMF14, and E. faecalis KGPMF47 were observed for lactose and mannitol. The results indicated the potential use of tested sugars and sugar substitutes as prebiotic compounds for the development of symbiotics with tested LAB, in medical or food industry.Publishe

In vitro interaction between Agrimonia eupatoria L. extracts and antibiotic

Synergistic activity between water, acetone, ethanol and diethyl ether extract of Agrimonia eupatoria L. and commonly used antibiotic (ampicillin) were evaluated. Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa were used. Interaction between plant extracts and antibiotic were tested by checkerboard method and expressed as fractional inhibitory concentration (FIC) index showed indifferent, additive and synergistic effects. Synergism was observed against E. coli for every combination of agents. FICI values were ranged from 0.03 to 0.29. Inhibitory concentration (IC50) was evaluated for every combination of tested extracts and antibiotic and the best combinations for every tested bacteria were combination of diethyl ether extract + ampicillin and combination of acetone extract + ampicillin

Broth depending production of extracellular enzymes by enterobacteria isolated from dairy food (Serbian cheese)

In this paper, we investigated, for the first time, the enzyme activity of enterobacteria from cheese from Southeastern Serbia (Sokobanja), which was produced in a traditional way. The tested bacteria were selected from the spring, summer, and autumn collections and grown in two different broths. The enzyme activity was investigated in the two broths and measured using spectrophotometry method. It was concluded that there were differences in the amount of extracellular enzymes depending on the broths. A statistically significant rise in the amount of enzymes was demonstrated in Tripton soy broth. Bacteria in cheese do not possess the ability to produce extracellular enzymes in large amounts. Therefore, they do not change the organoleptic characteristics of the cheese. There is a possibility that if they are found outside their natural environment (cheese), without interactions with antagonists, they may exhibit stronger enzyme activity.Publishe

The hydrophobicity of enterobacteria and their co-aggregation with Enterococcus faecalis isolated from Serbian cheese

In this paper, we investigated the hydrophobicity, ability to adhere to solvents and the pig epithelium and congregation of members of family Enterobacteriaceae and Enterococcus faecalis KGPMF 49. The bacteria used in this study were isolated from traditionally made autochthonous cheese from Southeastern Serbia (Sokobanja). The percentage of adhered bacteria was different in three solvents (chloroform, ethyl acetate and xylene). The highest percentage was detected in the presence of chloroform, and the lowest percentage was detected in the presence of xylene (chloroform < ethyl acetate < xylene). A different degree of co-aggregation of enterobacteria with E. faecalis KGPMF 49 was observed. Klebsiella ornithinolytica KGPMF 8 demonstrated the highest percentage of congregation with E. faecalis KGPMF49 (32.29%). Klebsiella pneumoniae KGPMF 13, K. ornithinolytica KGPMF 9 and Serratia marcescens biogp 1 KGPMF 19 were found to have the ability to adhere to the pig epithelium, whereas Escherichia coli KGPMF 22 showed no such ability. The ability to co-aggregate with other species and the ability to adhere to the pig epithelium are very important characteristics of the isolated bacteria.Publishe

In vitro evaluation of antimicrobial and antibiofilm activity of Oleum Hyperici: An original product from Goč Mountain (Serbia)

This study observed the preliminary investigation of Oleum Hyperici, an original product made from Hypericum perforatum flowers, collected from Goč Mountain (Serbia). Some of the chemical properties (pH, the content of metals and metalloids - As, Cr, Pb, Cd, Ni, Hg) and the microbiological safety of the product including the total count of aerobic mesophilic bacteria, coagulase positive staphylococci, Escherichia coli, Pseudomonas aeruginosa, Proteus sp., as well as fungi: molds and yeasts) were evaluated. Antimicrobial activity of H. perforatum oil against 18 species of bacteria was determined using the disc diffusion and microdilution methods, by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The antibiofilm activity of Oleum Hyperici was evaluated using the biofilm formation assay. The results indicated that oil did not contain potentially pathogenic microorganisms. Oleum Hyperici product showed the antimicrobial activity on the most of tested bacteria (inhibition zone was in a range from 14 - 24 mm), except on Klebsiella pneumoniae ATCC 70063, K. pneumoniae, E. coli ATCC 25922, P. mirabilis, Bacillus subtilis ATCC 6633 and B. subtilis IP 5832. According to the obtained results, tested bacteria (planktonic and biofilm form) showed sensitivity up to 10-2 dilution of Oleum Hyperici. Based on the obtained preliminary results, it could be concluded that Oleum Hyperici can be used in traditional medicine for development control of some potential human pathogens.Publishe

The simbiotic effect of temperature and sugars on the planktonic growth and biofilm formation of Klebsiella spp. isolated from traditionally made cheese

In this paper, we investigated the influence of temperatures (4°C, 37°C, 44°C) and different concentrations of glucose and lactose (0.5%, 1.5%, 2.5%, 3.5%) on the planktonic growth, biofilm formation and formed biofilm of Klebsiella oxytoca, Klebsiella ornithinolytica and Klebsiella pneumonia in two different broths (TSB and MH). The bacteria were isolated from the cheese produced in Southeastern Serbia. Klebsiella pneumoniae ATCC 70063 was used as a positive control. The planktonic growth and biofilm formation were measured using spectrophotometric method. Different broths caused a different planktonic growth and biofilm formation. At 4°C, the planktonic growth was not observed, neither was the formation of biofilm. Temperatures of 37°C and 44°C, as well as various concentrations of glucose and lactose, stimulated the planktonic growth of bacteria. The biofilm formation was less affected by glucose, but lactose stimulated the biofilm formation. The results of these were in accordance with the origin of bacteria, since the isolates were obtained from cheese. The intensity of the effect of sugars on the planktonic growth and biofilm formation depended on the type of bacteria.Publishe

Detection of enzymes produced by lactic acid bacteria isolated from traditionally made Serbian cheese and their role in the formation of its specific flavor

Nine species (sixteen isolates) of lactic acid bacteria (LAB) isolated from traditionally made Serbian cheese were evaluated for their enzymatic activities in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected based on their previously determined biochemical and physiological characteristics, as well as their antimicrobial activity, and were identified as Lactococcus lactis subsp. lactis (one isolate), Lc. lactis subsp. lactis biovar. diacetylactis (five isolates), Lactobacillus fermentum (two isolates), Lb. plantarum (one isolate), Lb. brevis (one isolate), Enterococcus faecalis (three isolates), E. faecium (one isolate), E. durans (one isolate) and E. hirae (one isolate). The enzymatic activities (acid and alkaline invertases, alkaline phosphatase, alkaline protease, a-amylase) were measured by using the spectrophotometric method. The results indicated that all Lactobacillus isolates showed protease, amylase, and alkaline phosphatase activities, while the activities of acid and alkaline invertases were not observed. The Lactococcus isolates showed protease, acid invertase and alkaline phosphatase activities, except the KGPMF50 isolate, which showed no alkaline phosphatase activity. The tested Enterococcus isolates showed weakly and strain-specific enzymatic activity. The results indicated that the enzymes produced by the investigated strains have a role in the formation of the specific flavor of cheese and that these isolates, especially Lactobacillus isolates, showed the potential for use in the dairy industry or applied biotechnology.Publishe