Outgrowth of spoilage yeasts and moulds and post-processing acidification can limit the shelf-life of some fermented dairy products including fresh lactic curd cheeses. The possibility of using high pressure processing (HPP) for controlling these problems was investigated in a commercially manufactured fresh lactic curd cheese (pH 4.3-4.4) and fermented milk models (pH 4.3-6.5). The effects of HPP at 300 and 600 MPa on inactivation of glycolytic enzymes of lactic acid bacteria were also evaluated. Fresh cheeses made from pasteurised bovine milk using a commercial Lactococcus starter preparation were treated with high pressures ranging from 200 to 600 MPa (less than or equal to 22°C, 5 min) under vacuum packaging conditions and subsequently stored at 4°C for 8 weeks. Treatment at greater than or equal to 300 MPa substantially reduced the viable count of Lactococcus and effectively prevented the outgrowth of yeasts and moulds for 6 to 8 weeks without adversely affecting the sensory and textural attributes of the product. However, it had no significant effects (p less than 0.01) on variation of titratable acidity during storage. Fermented milk models were prepared by individually growing Lactococcus lactis subsp. lactis C10, Lactococcus lactis subsp. cremoris BK5, Streptococcus thermophilus TS1, Lactobacillus acidophilus 2400 and Lactobacillus delbrueckii subsp. bulgaricus 2517 in UHT skim milk and diluting the resulting fermented milk with UHT skim milk up to pH 6.5. Pressure treatment of the milk models at pH 5.2 resulted in substantial inhibition of post-processing acidification during storage and markedly reduced the viable count of Lactococcus at both 300 and 600 MPa and other bacteria only at 600 MPa. Treatment of the milk model at 600 MPa decreased the viable counts of Candida zeylanoides and Candida lipolytica (wildtype spoilage yeasts of lactic curd cheese, added as challenge cultures) from 105 CFU mL-1 to below the detection limit (log 0 CFU mL-1) at all pH levels tested (pH 4.3-6.5) and effectively controlled their outgrowth for 8 weeks. Treatment of milk model at 300 MPa had a similar effect only on C. zeylanoides. The viable count of C. lipolytica was reduced by 2.6, 2.4 and 2.3 logs by treatment at 300 MPa at pH levels of 4.3, 5.2 and 6.5, respectively, which subsequently recovered by 2.9, 2.8 and 3.2 logs within 3 weeks. Glycolytic enzymes of various starter bacteria showed different responses to pressure treatment. The lactate dehydrogenase in L. lactis subsp. lactis and Lb. acidophilus was quite resistant to pressures up to 600 MPa, but it was almost completely inactivated in S. thermophilus at pressure levels as low as 300 MPa. The â-galactosidase in Lb. acidophilus was more pressure stable than â-galactosidase in S. thermophilus and Phospho-â-galactosidase in L. lactis subsp. lactis. The findings of this study suggests HPP at 300-600 MPa as an effective method for controlling the outgrowth of some spoilage yeasts and moulds in fresh lactic curd cheeses. The results obtained with selected lactic acid bacteria in fermented milk models can be used to assist in establishing HPP operating parameters for development of new generation cultured dairy products, of reduced acidity and extended shelf-life.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(農学) / 甲第19046号 / 農博第2124号 / 新制||農||1032 / 31997 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 加納 健司, 教授 植田 充美 / 学位規則第4条第1項該当

D. Tech. Food Technology. / Discusses a range of lactobacilli and bifidobacteria were isolated from functional food products and pharmaceutical preparations and also obtained from culture collections. They were then subjected to phylogenetic analysis for accurate identification and classification and the probiotic properties of the organisms was evaluated. The isolates were then screened for inhibitory activity against a range of pathogenic bacteria and Candida albicans strains. Selected isolates that were found to have the necessary inhibitory and probiotic properties were recommended for inclusion in an envisaged synbiotic, maize-based beverage that would, in a subsequent study, be subjected to a nutritional intervention trial aimed at alleviating oral thrush in human patients. The specific objectives: to investigate and illustrate the superiority of rpoA and pheS gene sequencing compared to 16S rRNA gene sequencing in the identification and phylogenic assignment of Lactobacillus isolates ; to determine the precision of selected protein-coding gene sequencing in comparison with 16S rRNA gene sequencing for the discrimination and phylogenetic analysis of Bifidobacterium isolates ; to investigate the probiotic properties of selected bacterial strains in terms of antibacterial activity, anti-Candida activity, acid resistance, bile tolerance and antibiotic resistance ; to determine the potential of Lactobacillus isolates to inhibit the growth of each of seven Candida albicans strains in fermented maize gruel and to establish the factors contributing to Candida inhibition and to determine the antimicrobial and antioxidant activity of intracellular extracts and to elucidate compounds in methanol extracts from selected Lactobacillus strains.

Lactic acid bacteria produce pediocin-like bacteriocins designated as Class Ha. These antimicrobial peptides are antagonistic against Listeria monocytogenes and other closely related Gram-positive bacteria Self-protection of the producer organism is attributed to the immunity proteins, encoded by genes that are eo-transcribed with the structural gene that encode the bacteriocin. The lactic acid bacterium, Leuconostoc gelidum UAL 187-22 is immune to its own bacteriocin, leucocin A. This is accredited to its immunity protein and the possible absence of a receptor on its cytoplasmic membrane. Leucocin A was purified from the supernatant of 1. gelidum to 90% purity by ion-exhange chromatography and C18 reverse phase High Pressure Liquid Chromatography (RP-HPLC) eluted with an acetonitrile, 0.1% Triflouroacetic acid (TFA) gradient. The immunity gene was isolated from the same producer using the polymerase chain reaction from the recombinant plasmid pJF 5.5 using primers EAL-2 and EAL-3. The amplicon was truncated into versions A and B by removing the C- and N-terminals, with HaeIII and ClaI restriction enzymes, respectively. The amplicon and the truncated fragments A and B were cloned into pMALc2 to construct recombinant plasmids pKPl, pKPIA and pKPIB, correspondingly, which were transformed into Escherichia coli (E. coli) strain JMI03. Clones were confirmed by colony PCR and Southern blot hybridization. The recombinant clones were subsequently expressed as MBP-IP, MBP-IPA and MBP-IPB fusion proteins that were verified by Western blot using the anti-MBP antibody. Factor Xa protease was used to cleave MBP from the proteins of interest. The resulting pure immunity protein versions had an approximate molecular weight of slightly more that 10 kDa. The binding interactions of the purified immunity protein constructs and leucocin A were compared on the Biacore 2000 instrument with surface plasmon resonance. None of the immunity constructs interacted with leucocin A, however, the N-terminal region of the immunity protein interacted with the cytoplasmic extract. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

To gain a better understanding of contaminating bacteria in bioethanol industry, we profiled the bacterial community structure in corn-based bioethanol fermentations and evaluated its correlation to environmental variables. Twenty-three batches of corn-mash sample were collected from six bioethanol facilities. The V4 region of the collective bacterial 16S rRNA genes was analyzed by Illumina Miseq sequencing to investigate the bacterial community structure. Non-metric multidimensional scaling (NMDS) ordination plots were constructed to visualize bacterial community structure groupings among different samples, as well as the effects of multiple environmental variables on community structure variation. Our results suggest that bacterial community structure is facility-specific, although there are two core bacterial phyla, Firmicutes and Proteobacteria. Feedstock, facility, and fermentation technology may explain the difference in community structure between different facilities. Lactic acid, the most important environmental variable that influences bacterial community structure grouping, could be utilized as an indicator of bacterial contamination. We also identified genes responsible for the multiple antibiotic-resistance phenotype of an Enterobacter cloacae strain isolated from a bioethanol fermentation facility. We performed PCR assays and revealed the presence of canonical genes encoding resistance to penicillin and erythromycin. However, a gene encoding resistance to virginiamycin was not detected.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2006. / Härtill 4 uppsatser.

Thesis (Ph.D.)--Mississippi State University. Department of Food Science, Nutrition and Health Promotion. / Title from title screen. Includes bibliographical references.

Thesis (MSc Food Sc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: A wide variety of traditionally and commercially fermented milks are commonly consumed in various countries of Sub-Saharan Africa. Commercially fermented milk is produced on an industrial scale according to well-managed, standardised production processes and starters are used to initiate fermentation. Traditionally fermented milk is prepared domestically and fermentation occurs spontaneously at ambient temperatures. Lactic acid bacteria (LAB) are responsible for milk fermentation during which they convert the milk carbohydrates to lactic acid, carbon dioxide, alcohol and other organic metabolites. Acetic acid bacteria (AAB), yeasts and mycelial fungi have also been isolated from fermented milks. In this study the microbial consortium present in three traditionally fermented milks, namely omashikwa from Namibia, masse from Mozambique and chekapmkaika from Uganda and two commercially fermented milks, namely chambiko from Malawi and omaere from Namibia, were isolated and enumerated on six different selective media that included MSR + C (specific for lactobacilli), KCA + TTC (specific for lactococci), KCA + V (specific for leuconostocs), MRS + E (specific for AAB), MEA (specific for mycelial fungi) and YPD (specific for yeasts). No significant differences were found between the enumeration values obtained for the three chambiko samples, as well as for enumeration values obtained for the two omaere samples on each of the selective media, indicating low sample variance. Significant differences between enumeration values obtained for the three omashikwa samples were found on all six selective media. Significant differences between enumeration values of the three masse samples and both the chekapmkaika samples were also observed on the selective media. In addition to this, significant differences were observed between average enumeration values obtained for each media between the masse and chekapmkaika, the chambiko and omaere, as well as when the traditional and commercial milks were compared. According to the average enumeration values obtained on each media selective for LAB, the highest bacterial counts were detected on KCA + TTC medium for omaere (2.3 x 106 cfu.ml-1), KCA + V for chambiko (1.8 x 105 cfu.ml-1), KCA + TTC for omashikwa and MRS + C for masse and chekapmkaika (6.2 x 106 and 2.0 x 103 cfu.ml-1, respectively). After isolation and enumeration of the microbes present in each milk, bacterial isolates on the media selective for LAB and AAB were obtained according to the Harrison Disk method. These isolates were identified by amplifying a 1.5 kilobase (kb) part of the 16S ribosomal RNA (rRNA) gene using the polymerase chain reaction (PCR), followed by DNA sequencing. The isolates were identified by comparing the sequences obtained to sequences listed in the NCBI database using the BLAST algorithm and searching for the closest relative. The main LAB group present in the omaere was lactococci (94%), in chambiko and chekapmkaika it was lactobacilli (30% and 45%, respectively), in omashikwa it was enterococci (43%) and in masse it was leuconostocs (68%). The same microbial species were present on a number of the selective media used in this study. Lactococcus spp., Enterococcus spp. and Lactobacillus spp. were isolated from MRS + C, KCA + TTC, KCA + V and MRS + E and Leuconostoc spp. were isolated from MRS + C, MRS + E and KCA + V. Hygienic standards during traditional milk fermentation is often poor and, therefore, microbial contaminants were isolated from the traditional milk and these included Acinetobacter johnsonii and Klebsiella pneumoniae from KCA + V, Mesorhizobium loti, Acinetobacter radioresistens, Escherichia coli, Staphylococcus spp., Kluyvera georgiana, Enterobacter spp. and Klebsiella oxytoca from KCA + TTC, Staphylococcus spp. from MRS + C and Bacillus spp. from MRS + E. Since the media used for the isolation of the LAB and AAB in this study were not selective further identification of the enumerated microbes is of importance for the identification of the microbial groups present in each fermented milk. The data obtained in this study clearly shows that fermented milks from Sub-Saharan Africa vary significantly from each other in terms of microbial numbers, microbial diversity and the dominant microbial groups present. The microbial diversity of the traditionally fermented milks was more diverse than the microbial diversity of the commercially fermented milks. LAB strains isolated from these traditionally fermented milks can be used to develop novel starters and as a result new commercially fermented dairy products with unique aromas, tastes and characteristics can be produced. / AFRIKAANSE OPSOMMING: 'n Wye verskeidenheid tradisioneel en kommersieel gefermenteerde melk produkte word algeneem verbruik in verskeie lande van Sub-Sahara Afrika. Kommersieel gefermenteerde melk word geproduseer op groot skaal, deur deeglik bestuurde gestandardiseerde produksieprosesse en 'n beginkultuur word gebruik om fermentasie te inisieer. Tradisioneel gefermenteerde melk word tuis gemaak en fermentasie gebeur spontaan by kamertemperatuur. Melksuurbakterieë (MSB) is verantwoordelik vir melkfermentasie waartydens die bakterieë koolhidrate omskakel na melksuur, koolstofdioksied, alkohol en ander organiese sure. Asetaatsuurbakterieë (ASB), giste en miseliale fungi is ook al van gefermenteerde melk geïsoleer. In hierdie studie is die mikrobiese konsortium teenwoordig in drie soorte tradisioneel gefermenteerde melk, naamlik omashikwa van Namibië, masse van Mosambiek en chekapmkaika van Uganda en twee soorte kommersieel gefermenteerde melk, naamlik chambiko van Malawi en omaere van Namibië, geïsoleer en getel op ses verskillende selektiewe groeimedia insluitend MRS + C (spesifiek vir lactobacilli), KCA + TTC (spesifiek vir lactococci), KCA + V (spesifiek vir leuconostocs), MRS + E (spesifiek vir ASB), MEA (spesifiek vir miseliale fungi) en YPD (spesifiek vir giste). Geen betekenisvolle verskille is gevind tussen die mikrobiese tellings verkry vir die drie chambiko monsters nie, sowel as tussen die mikrobiese tellings verkry vir die twee omaere monsters, op elk van die selektiewe groeimedia, wat dui op lae monster variansie. Betekenisvolle verskille is gevind tussen die mikrobiese tellings verkry vir die drie omashikwa monsters op al ses selektiewe groeimedia. Betekenisvolle verskille is ook waargeneem tussen die mikrobiese tellings van die drie masse monsters en beide die chekapmkaika monsters op die selektiewe groeimedia. Daarbenewens is betekenisvolle verskille waargeneem tussen gemiddelde mikrobiese tellings verkry vir elke groeimedium tussen die masse en chekapmkaika, die chambiko en omaere asook toe die tradisionele en kommersiële melk produkte met mekaar vergelyk is. Volgens die gemiddelde mikrobiese tellings verkry op elk van die groeimedia selektief vir MSB, is die hoogste mikrobiese telling waargeneem op KCA + TTC medium vir omaere (2.3 x 106 kve.ml-1), KCA + V vir chambiko (1.8 x 105 kve.ml-1), KCA + TTC vir omashikwa en MRS + C vir masse en chekapmkaika (6.2 x 106 en 2.0 x 103 kve.ml-1, respektiewelik). Na die isolasie en tel van die mikrobes teenwoordig in elke melk is bakteriese isolate op die media selektief vir MSB en ASB verkry volgends die Harrison Disk metode. Hierdie isolate is geïdentifiseer deur amplifikasie van „n 1.5 kilobasis (kb) gedeelte van die 16S ribosomale RNS (rRNS) geen deur gebruik te maak van die polimerase kettingreaksie gevolg deur DNS klonering. Die isolate is geïdentifiseer deur die gekloneerde insetsels se volgordes te vergelyk met volgordes beskikbaar op die NCBI webwerf deur van die BLAST algoritme gebruik te maak en die naas verwante insetsel op te spoor. Die hoof MSB groep teenwoordig in die omaere was lactococci (94%), in chambiko en chekapmkaika was dit lactobacilli (30% en 45%, respektiewelik), in die omashikwa was dit enterococci (43%) en in die masse was dit leuconostocs (68%). Dieselfde mikrobiese spesies was teenwoordig op verskeie van die selektiewe groeimedia gebruik in hierdie studie. Lactococcus spp., Enterococcus spp. en Lactobacillus spp. is geïsoleer van MRS + C, KCA + TTC, KCA + V en MRS + E en Leuconostoc spp. is geïsoleer van MRS + C, MRS + E en KCA + V. Higiëniese standaarde tydens tradisionele melkfermentasie is dikwels swak en dus is mikrobiese kontaminante geïsoleer van die tradisionele melk produkte insluitend Acinetobacter johnsonii en Klebsiella pneumoniae van KCA + V, Mesorhizobium loti, Acinetobacter radioresistens, Escherichia coli, Staphylococcus spp., Kluyvera georgiana, Enterobacter spp. en Klebsiella oxytoca van KCA + TTC, Staphylococcus spp. van MRS + C en Bacillus spp. van MRS + E. Aangesien die media wat gebruik is vir die isolasie van die MSB en ASB in hierdie studie nie selektief was nie, is verdere identifikasie van die getelde mikrobes belangrik vir die identifikasie van die mikrobiese groepe teenwoordig in elke melk. Die data verkry in hierdie studie dui aan dat gefermenteerde melk produkte van Sub-Sahara Afrika betekenisvol van mekaar verskil in terme van mikrobiese getalle, mikrobiese diversiteit en die dominante mikrobiese groepe teenwoordig. Die mikrobiese diversiteit van die tradisioneel gefermenteerde melk produkte was meer divers as die mikrobiese diversiteit van die kommersieel gefermenteerde melk produkte. MSB spesies geïsoleer van hierdie tradisioneel gefermenteerde melk produkte kan gebruik word om nuwe beginkulture te ontwikkel en gevolglik kan nuwe kommersieel gefermenteerde suiwelprodukte met unieke aromas, smake en eienskappe geproduseer word.