Leuconostoc spoilage of refrigerated, packaged foods

Leuconostoc spp. are lactic acid bacteria (LAB) implicated in food spoilage, especially on refrigerated, modified atmosphere packaged (MAP) meats. The overall aim of this thesis was to learn more about Leuconostoc spp. as food spoilage organisms with a focus on commercial products where LAB spoilage is considered a problem and the main factor limiting shelf-life. Therefore, we aimed to identify Leuconostoc spp. involved in food spoilage, as well as to characterise the spoilage reactions they caused and their contamination sources during poultry meat processing. In addition, we examined the distribution of strains of Leuconostoc gasicomitatum in different food commodities. Finally, we analysed the genome content of L. gasicomitatum LMG 18811 with a special focus on metabolic pathways related to food spoilage. The findings show that Leuconostoc gelidum and L.gasicomitatum were responsible for the discoloration and off-odours developed in beef steaks. Together with Leuconostoc mesenteroides, these Leuconostoc spp., also cause spoilage of vegetable sausages. In contrast, we showed that Leuconostoc spp. are not important for the shelf-life or quality of non-marinated broiler products although, in marinated broiler fillet products, Leuconostoc spp., L.gasicomitatum in particular, are considered spoilage organisms. Furthermore, the findings of the contamination survey we carried out in a poultry processing plant indicated that spoilage Leuconostoc spp. are derived from the processing environment rather than from the broilers, and that air movement distributes psychrotrophic spoilage LAB, including leuconostocs, and has an important role in meat contamination during poultry processing. Pulsed-field gel electrophoresis (PFGE) based genotyping of L. gasicomitatum strains demonstrated that certain genotypes are common in various meat products. In contrast, genotypes associated with meat were not recovered in vegetable-based sources. This suggests that these two food categories either become contaminated with, or favour the growth of different genotypes. Furthermore, the results indicated that the meat processing environment contributes to L. gasicomitatum contamination as certain genotypes were repeatedly identified from products of the same processing plant. Finally, the sequenced and annotated genome of L.gasicomitatum LMG 18811 allowed us to identify the metabolic pathways and reactions resulting in food spoilage.

Temperature effect on dextransucrase production by Leuconostoc mesenteroides FT 045 B isolated from Alcohol and Sugar Mill Plant

Temperature (23 to 31 degrees C) and sucrose concentration ( 3 and 4%) effects on dextransucrase production by Leuconostoc mesenteroides NRRL B 512 ( F) and Leuconostoc mesenteroides FT 045 B were studied. The conditions in all fermentations were: total reaction volume 2 L, 132 rev. min-1, 0.5 vvm and pH 6.0. The optimum temperature for enzyme yield for strain NRRL B 512 ( F) was 23 degrees C, where at 8-h fermentation was possible to achieve 49.3 DSU/mL. When FT 045 B strain was utilized, 3.2 DSU/mL was obtained at temperature 23 to 25 degrees C.

d(-)-Lactic Acid Production by Leuconostoc mesenteroides B512 Using Different Carbon and Nitrogen Sources

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structural characterization of a new dextran with a low degree of branching produced by Leuconostoc mesenteroides FT045B dextransucrase

This paper offers the physical and chemical characterization of a new dextran produced by Leuconostoc mesenteroides FT045B. The chemical structure was determined by Fourier Transform Infrared spectroscopy and 1H Nuclear Magnetic Resonance spectroscopy. The dextran was hydrolyzed by endodextranase; the products were analyzed using thin layer chromatography and compared with those of commercial B-512F dextran. The number-average molecular weight and degree of polymerization of the FT045B dextran were determined by the measurement of the reducing value using the copper bicinchoninate method and the measurement of total carbohydrate using the phenol-sulfuric acid method. The data revealed that the structure of the dextran synthesized by FT045B dextran sucrase is composed of d-glucose residues, containing 97.9% α-(1,6) linkages in the main chains and 2.1% α-(1,3) branch linkages compared with the commercial B-512F dextran, which has 95% α-(1,6) linkages in the main chains and 5% α-(1,3) branch linkages. © 2012 Elsevier Ltd. All rights reserved.

Estudo e otimização da produção da dextranasacarase e caracterização da dextrana produzida por Leuconostoc mesenteroides FT045B

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Estudo da produção de dextranasacarase por Leuconostoc mesenteroides FT 045 B

Pós-graduação em Ciências Biológicas (Microbiologia Aplicada) - IBRC

Leuconostoc mesenteroides SJRP55 isolada de mussarela de búfala: perfil probiótico e bacteriocinogênico

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Leuconostoc mesenteroides SJRP55: a potential probiotic strain isolated from Brazilian water buffalo mozzarella cheese

The probiotic potential of Leuconostoc mesenteroides subsp. mesenteroides SJRP55 isolated from water buffalo mozzarella cheese was evaluated. The microorganism presented resistance to stressful conditions that simulated the gastrointestinal tract, and to the best of our knowledge Leuconostoc mesenteroides SJRP55 was the first of this species with the ability to deconjugate bile salts. Tolerance to NaCl was temperature dependent, as well the results obtained by aggregation capacity. The strain presented good adhesion properties, β galactosidase activity, viability in fermented milk during storage, non-active against Streptococcus thermophilus and sensible to most of the tested antibiotics. Some analgesic medications inhibited the growth of the strain. Leuconostoc mesenteroides SJRP55 exhibited in vitro probiotic potential, and it can be better characterized through future in vivo tests. This bacterium presents higher functional properties compared to other studied strains, and therefore it is a potential candidate for the application as a probiotic strain, which could be used by industries in the manufacture of functional milk-based products.

Leuconostoc mesenteroides SJRP55: a bacteriocinogenic strain isolated from Brazilian water buffalo mozzarella cheese

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The two faces of leuconostoc mesenteroides in food systems

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cystine-mediated oxidative defence in Lactobacillus reuteri BR11

Lactobacillus reuteri BR11 possesses an abundant cystine uptake (Cyu) ABC-transporter that was previously found to be involved in a novel mechanism of oxidative defence mediated by cystine. The current study aimed to elucidate this mechanism with a focus on the role of the co-transcribed cystathionine ã-lyase (Cgl). Growth studies of wild-type L. reuteri BR11 and mutants inactivated in cgl and the cystine-binding protein encoding gene cyuC showed that in contrast to the Cyu transporter, whose inactivation led to growth arrest in aerated cultures, Cgl is not crucial for oxidative defence. However, the role of Cgl in oxidative defence became apparent in the presence of severe oxidative damage and cysteine deprivation. Cysteine was found to be protective against oxidative stress, and the action of Cgl in both cysteine biosynthesis and degradation poses a seemingly futile pathway that deprives the intracellular cysteine pool. To further characterise the relationship between Cgl activity and cysteine and their roles in oxidative defence, enzymatic assays were performed on purified Cgl, and intracellular concentrations of cysteine, cystathionine and methionine were determined. Cgl was highly active towards cystine and cystathionine and less active towards cysteine in vitro, suggesting the main function of Cgl to be cysteine biosynthesis. Cysteine was found at high concentrations in the cell, but the levels were not significantly affected by inactivation of cgl or growth under aerobic conditions. It was concluded that both anabolic and catabolic activities of Cgl towards cysteine contribute to oxidative defence, the former by maintaining an intracellular reservoir of thiol analogous to glutathione, and the latter by producing H2S which is readily secreted, thus creating a reducing extracellular environment. The significance of the Cyu transporter to the physiology of L. reuteri BR11 prompted a phylogenetic study to determine its presence in bacteria. Orthologs of the Cyu transporter that are closest matches to the Cyu transporter are only limited to several species of Lactobacillus and Leuconostoc. Outside the Lactobacillales order, the closest matching orthologs belong to Proteobacteria, and there are more orthologs in Proteobacteria than non-Lactobacillales Firmicutes, suggesting that the Cyu transporter locus was present in the ancestor of the Proteobacteria and Firmicutes, and over evolutionary time has been lost or diverged in many Firmicutes. The clustering of the Cyu transporter locus with a gene encoding a Cgl family protein is even rarer. It was only found in L. reuteri, Lactobacillus vaginalis, Weissella paramesenteroides, the Lactobacillus casei group, and several Campylobacter sp. An accompanying phylogenetic study of L. reuteri BR11 using multi-locus sequence analysis showed that L. reuteri BR11 had diverged from more than 100 strains of L. reuteri isolated from various hosts and geographical locations. However, comparison with other Lactobacillus species supported the current classification of BR11 as L. reuteri. The most closely related species to L. reuteri is L. vaginalis or Lactobacillus antri, depending on the housekeeping gene used for analysis. The close evolutionary relationship of L. vaginalis to L. reuteri and the high degree of sequence identity between the cgl-cyuABC loci in both species suggest that the Cyu system is highly likely to perform similar functions in L. vaginalis. In search of other genes that function in oxidative defence, a number of mutants which were inactivated in genes that confer increased resistance to oxidative stress in other bacteria were constructed. The genes targeted were ahpC (peroxidase component of the alkyl hydroperoxide reductase system), tpx (thiol peroxidase), osmC (osmotically induced protein C), mntH (Mn2+/Fe2+ transporter), gshA (ã-glutamylcysteine synthetase) and msrA (methionine sulfoxide reductase). The ahpC and mntH mutants had slightly lower minimum inhibitory concentrations of organic peroxides, suggesting these genes might be involved in resistance to organic peroxides in L. reuteri. However, none of the mutants exhibited growth defects in aerated cultures, in stark contrast to the cyuC mutant. This may be due to compensatory functions of other genes, a hypothesis which cannot be tested until a robust protocol for constructing markerless multiple gene deletion mutants in L. reuteri is developed. These results highlight the importance of the Cyu transporter in oxidative defence and provide a foundation for extending the research of this system in other bacteria.

Immunomodulatory effects of probiotic bacteria in healthy adults

Probiooteilla kantakohtaisia vaikutuksia ihmisen immuunijärjestelmään terveillä aikuisilla Probiooteilla on kantakohtaisia tulehduksen välittäjäaineita vähentäviä vaikutuksia ja probioottien yhdistelmien vaikutukset eroavat yksittäisten kantojen vaikutuksista selviää TtM Riina Kekkosen tuoreesta väitöstutkimuksesta. TtM Riina Kekkonen on selvittänyt väitöskirjassaan eri probioottikantojen vaikutuksia immuunivasteeseen valkosolumallissa sekä terveillä aikuisilla lumekontrolloiduissa kliinisissä tutkimuksissa. Aikaisemmin probioottien vaikutuksia on tutkittu lähinnä allergian ja erilaisten vatsavaivojen ehkäisyssä ja hoidossa. Probiootteja sisältäviä tuotteita käyttävät kuluttajat ovat kuitenkin useimmiten terveitä aikuisia, ja probioottien vaikutus terveiden aikuisten immuunijärjestelmään on ollut puutteellisesti selvitettyä. Valkosolumallissa probioottikantojen havaittiin poikkeavan toisistaan niiden kyvyssä aktivoida immuunivasteen välittäjäaineiden, sytokiinien, tuotantoa. Anti-inflammatorisia, eli tulehdusta lievittäviä vaikutuksia nähtiin lähinnä Bifidobacterium ja Propionibacterium sukuihin kuuluvilla kannoilla. Streptococcus ja Leuconostoc sukuihin kuuluvat kannat puolestaan aktivoivat Th1 tyyppistä, soluvälitteistä immuunivastetta. Eri probioottien kombinaatiot eivät saaneet aikaan voimakkaampaa aktivaatiota yksittäisiin kantoihin verrattuna, joka viittaa probioottien keskinäiseen kilpailuun niiden ollessa kontaktissa ihmisen solujen kanssa. Probioottikantojen valinta kliinisiin tutkimuksiin tehtiin niiden anti-inflammatoristen ominaisuuksien perusteella. Parhaita anti-inflammatorisia kantoja olivat B. lactis ssp. animalis Bb12 ja P. freudenreichii ssp. shermanii JS, joiden lisäksi tutkimuksiin valittiin myös L. rhamnosus GG (LGG) hyvin tutkittuna referenssikantana. Solutöiden tulokset eivät olleet täysin verrannollisia kliinisen työn tuloksiin, koska LGG näytti omaavan parhaat anti-inflammatoriset ominaisuudet kliinisissä tutkimuksissa vaikka solutyössä sen aikaansaamat vasteet olivat melko vaimeita. Kolmen viikon kliinisessä tutkimuksessa terveillä aikuisilla LGG alensi mm. tulehdusta kuvaavan C-reaktiivisen proteiinin ja inflammatoristen sytokiinien määrää. Pidemmässä kolmen kuukauden pituisessa kliinisessä tutkimuksessa LGG:llä ei ollut vaikutusta terveiden aikuisten infektiosairastavuuteen, mutta LGG lyhensi vatsavaivojen kestoa. Probioottien vaikutukset immuunijärjestelmään näyttävät olevan kantakohtaisia ja erityisesti Lactobacillus rhamnosus GG:llä havaittiin anti-inflammatorisia vaikutuksia. Valkosolumallia ei tulisi käyttää ainoana probioottikantojen skriinausmenetelmänä niiden immunologisia vaikutuksia selvitettäessä, koska solutöiden tulokset eivät olleet täysin verrannollisia kliinisten tutkimusten tuloksiin. Sen sijaan veren perifeeristen lymfosyyttien eristäminen ja niiden aktivoitumisen selvittäminen lyhytaikaisessa kliinisessä tutkimuksessa voisi toimia suhteellisen helppona skiinausmenetelmänä.

Äidinmaito - terveysjuomaa ja normaalibakteereita

The chemical composition of breast milk has been studied in detail in the past decades. Hundreds of new antibacterial and antiviral components have been found. Several molecules have been found to promote the proper function of neonatal intestine. However, microbiological studies of breast milk have been, until recently, focused mainly on detecting harmful and pathogenic bacteria and viruses. Natural microbial diversity of human milk has not been widely studied before the work reported in this thesis. This is mainly because breast milk has traditionally been thought to be sterile - even if a certain amount of commensal bacteria have usually been detected in milk samples. The first part of this licentiate thesis contains a short literature review about the anatomy and physiology of breast feeding, human milk chemical and microbiological composition, mastitis, intestinal flora and bacteriocins. The second part reports on the experiments of the licentiate work, concentrating on the microbial diversity in the milk of healthy breast-feeding mothers, and the ability of these bacteria to produce antibacterial substances against pathogenic bacteria. The results indicate that human milk is a source of commensal bacteria for infant intestine. 509 random isolates from 40 breast milk samples were isolated and identified by 16S rRNA sequencing. Median bacterial count was about 600 colony forming units per milliliter. Over half of the isolates were staphylococci, and almost one third streptococci. The most common species were skin bacteria Staphylococcus epidermidis and oral bacteria Streptococcus salivarius and Streptococcus mitis. Lactic acid bacteria, identified as members of Lactobacillus-, Lactococcus- and Leuconostoc -genera, were found in five milk samples. Enterococci were found in three samples. A novel finding in this study is the capability of these commensal bacteria to inhibit the growth of pathogens. In 90 precent of the milk samples commensal bacteria inhibiting the growth of Staphylococcus aureus were found. In 40 precent of samples the colonies could block the growth completely. One fifth of the isolated Staph. epidermidis strains, half of Str. salivarius strains, and all lactic acid bacteria and enterococci could inhibit or block the growth of Staph. aureus. In further study also Listeria innocua- and Micrococcus luteus active isolates were found in 33 and 11 precent of milk samples (out of 140). Furthermore, two Lactococcus lactis isolates from the breast milk were shown to produce bacteriocin nisin, which is an antimicrobial molecule used as a food preservative. The importance of these human milk commensal bacteria in the development of newborn intestinal flora and immune system, as well as in preventing maternal breast infections, should be further explored.

Viilin varahapatteen kehittäminen

Kirjallisuuskatsauksessa käsiteltiin viiliä hapanmaitotuotteena, viilin arviointimenetelmiä sekä viilihapatteissa käytettyjen hapatekantojen vaikutuksia maidossa ja niiden vuorovaikutuksia toisiinsa. Lisäksi pohdittiin viilihapatteiden bakteriofaageja, niiden ehkäisyä sekä solunsisäisiä että -ulkoisia faagiresistenssimekanismeja. Kokeellisessa osassa tutkittiin, onko nykyisten tuotantohapatteiden koostaminen yksittäiskannoista mahdollista, sekä koostettiin uusista Lactococcus lactis ssp. cremoris -kannoista varahapate. Hapatteiden käyttökelpoisuutta tutkittiin rakennemittauksin ja aistinvaraisin menetelmin. Varahapatteen faagikestävyyttä testattiin valmistamalla viiliä ja infektoimalla viilit faaginäytteillä. Hapatekannat viljeltiin fermentorissa, konsentroitiin sentrifugoimalla ja pakastettiin –75 °C:ssa. Hapatteet koostettiin noin 1 päivä ennen viilin valmistusta. Viilit arvioitiin aistinvaraisesti 3–6 hengen ryhmässä ja viileille tehtiin rakennemittaukset (kiinteys, sakeus ja koossapysyvyys) sekä kemialliset analyysit. Aistinvaraisen arvioinnin tulokset käsiteltiin tilastollisesti ja tulosten perusteella tehtiin uudet kantakombinaatiot ja viilit. Tuotantohapatekannoilla valmistetut viilit arvioitiin kolmitestillä (n = 10–11) ja uusilla kannoilla valmistetut viilit arvioitiin profiilitestillä (n = 8). Lisäksi viileille tehtiin rakennemittaukset ja kemialliset analyysit. Varahapatekoosteilla valmistettujen viilien pH laski 4,5:een faagin läsnäollessa 0–10 tunnin viiveellä verrattuna faagivapaisiin viileihin, kun taas tuotantohapatteet eivät hapantuneet faagin läsnäollessa. Aromintuottajat eivät kasvaneet viileissä kunnolla, kun hapate koostettiin yksittäiskannoista. Kolmitestissä ei erotettu nykyisillä tuotantohapatteilla valmistettua viiliä yksittäin koostetusta hapatteesta, eli hapatteita on mahdollista koostaa yksittäiskannoista. Varahapatteilla koostetut viilit poikkesivat profiilitestissä tuotantohapatteella koostetusta viilistä ulkonäkö- ja rakenneominaisuuksiltaan. Makuominaisuuksien suhteen ei viilien välille saatu eroa.

Study on effects of extracted probiotics from microbial flora of digestive system on growth indices in beluga (Huso huso) and Persian sturgeon (Acipenser persicus)

Isolation of Lactic Acid Bacteria (LAB) was carried out from gastro intestinal tract of beluga and Persian sturgeon at international sturgeon research institute and PCR has been used for bacteria Identification. Two species of LAB including Enterococcus seriolicida and Leuconostoc mesenteroides were isolated from Gastrointestinal tract (GI) of persian sturgeon in this study and the counts of Leu. mesenteroides (4.63×102 CFU/gr of GI) was significantly higher than other species. Lactobacillus curvatus, Lactococcus raffinolactis, Lactococcus lactis and Streptococcus sp. were also isolated from GI of beluga and maximum counts was belonged to Lb. curvatus (4.63×102 CFU/gr of GI) in this species. Dominant species were lyophilized and adding to the water since start of mix feeding of sturgeon with different counts including 2×109, 5×109 and 9×109 CFU/gr of live food, 4 times a day. The results revealed that the maximum and minimum growth rate and protease, amylase, and lipase activity in beluga was gained by using of Lb. curvatus with total viable count of 9×10 9 CFU/gr of live food and Leu. mesenteroides with total viable count of 9×109 CFU/gr of live food. According to the results of this study, the maximum and minimum growth rate and protease, amylase, and lipase activity in Persian sturgeon was gained by using of Leu. mesenteroides with total viable count of 2×10 9 CFU/gr of live food and Lb. curvatus with total viable count of 9×109 CFU/gr of live food. Histological study showed that gastrointestinal development was same during larva rearing in control and other treatments but the size of liver was bigger in treatments that received nonspecific LAB in both species. According to the results, positive effects of using dominant specific LAB bacteria for larviculture of sturgeon has been proved in this study.