Direct fermentation of potato starch in wastewater to lactic acid by Rhizopus oryzae

The fungal species of Rhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater. Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L at pH 6.0 and 30degreesC was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%similar to85.5% associated with 1.5similar to2.0 g/L fungal biomass produced in 36 h of fermentation.

Direct fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of direct fermentation for lactic acid production by fungal species of Rhizopus arrhizus 3,6017 and Rhizopus oryzae 2,062 was studied with respect to growth pH, temperature and substrate. The direct fermentation was characterized by starch hydrolysis, accumulation of reducing sugar, and production of lactic acid and fungal biomass. Starch hydrolysis, reducing sugar accumulation, biomass formation and lactic acid production were affected with the variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30 degrees C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.87-0.97 g/g starch associated with 1.5-2.0 g/l fungal biomass produced in 36 h fermentation. R. arrhizus 3,6017 had a higher capacity to produce lactic acid, while R. oryzae 2,062 produced more fungal biomass under similar conditions.

Simultaneous saccharification and fermentation of potato starch wastewater to lactic acid by Rhizopus oryzae and Rhizopus arrhizus

The biochemical kinetic of simultaneous saccharification and fermentation (SSF) for lactic acid production by fungal species of Rhizopus arrhizus 36017 and Rhizopus oryzae 2062 was studied with respect to growth pH, temperature and substrate. Both R. arrhizus 36017 and R. oryzae 2062 had a capacity to carry out a single stage SSF process for lactic acid production from potato starch wastewater. The kinetic characteristics, termed as starch hydrolysis, accumulation of reducing sugars, lactic acid production and fungal biomass formation, were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/l at pH 6.0 and 30 degrees C was favourable for both starch saccharification and lactic acid fermentation, resulting in lactic acid yield of 0.85-0.92 g/g associated with 1.5-3.5 g/l fungal biomass produced in 36-48 h fermentation. R. arrhizus 36017 had a higher capacity to produce lactic acid, while R. oryzae 2062 produced more fungal biomass under similar conditions. (c) 2005 Elsevier B.V. All rights reserved.

Survival of probiotics encapsulated in chitosan-coated alginate beads in yoghurt from UHT- and conventionally treated milk during storage

Survival of the microencapsulated probiotics, Lactobacillus acidophilus 547, Bifidobacterium bifidum ATCC 1994, and Lactobacillus casei 01, in stirred yoghurt from UHT- and conventionally treated milk during low temperature storage was investigated. The probiotic cells both as free cells and microencapsulated cells (in alginate beads coated with chitosan) were added into 20 g/100 g total solids stirred yoghurt from UHT-treated milk and 16 g/100 g total solids yoghurt from conventionally treated milk after 3.5 h of fermentation. The products were kept at 4 degrees C for 4 weeks. The survival of encapsulated probiotic bacteria was higher than free cells by approximately 1 log cycle. The number of probiotic bacteria was maintained above the recommended therapeutic minimum (10(7) cfu g(-1)) throughout the storage except for R bifidum. The viabilities of probiotic bacteria in yoghurts from both UHT- and conventionally treated milks were not significantly (P > 0.05) different. (c) 2004 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Biodiversity of the surface microbial consortia from Limburger, Reblochon, Livarot, Tilsit, and Gubbeen cheeses

Comprehensive collaborative studies from our laboratories reveal the extensive biodiversity of the microflora of the surfaces of smear-ripened cheeses. Two thousand five hundred ninety-seven strains of bacteria and 2,446 strains of yeasts from the surface of the smear-ripened cheeses Limburger, Reblochon, Livarot, Tilsit, and Gubbeen, isolated at three or four times during ripening, were identified; 55 species of bacteria and 30 species of yeast were found. The microfloras of the five cheeses showed many similarities but also many differences and interbatch variation. Very few of the commercial smear microorganisms, deliberately inoculated onto the cheese surface, were reisolated and then mainly from the initial stages of ripening, implying that smear cheese production units must have an adventitious "house" flora. Limburger cheese had the simplest microflora, containing two yeasts, Debaryomyces hansenii and Geotrichum candidum, and two bacteria, Arthrobacter arilaitensis and Brevibacterium aurantiacum. The microflora of Livarot was the most complicated, comprising 10 yeasts and 38 bacteria, including many gram-negative organisms. Reblochon also had a very diverse microflora containing 8 yeasts and 13 bacteria (excluding gram-negative organisms which were not identified), while Gubbeen had 7 yeasts and 18 bacteria and Tilsit had 5 yeasts and 9 bacteria. D. hansenii was by far the dominant yeast, followed in order by G. candidum, Candida catenulata, and Kluyveromyces lactis. B. aurantiacum was the dominant bacterium and was found in every batch of the 5 cheeses. The next most common bacteria, in order, were Staphylococcus saprophyticus, A. arilaitensis, Corynebacterium casei, Corynebacterium variabile, and Microbacterium gubbeenense. S. saprophyticus was mainly found in Gubbeen, and A. arilaitensis was found in all cheeses but not in every batch. C. casei was found in most batches of Reblochon, Livarot, Tilsit, and Gubbeen. C. variabile was found in all batches of Gubbeen and Reblochon but in only one batch of Tilsit and in no batch of Limburger or Livarot. Other bacteria were isolated in low numbers from each of the cheeses, suggesting that each of the 5 cheeses has a unique microflora. In Gubbeen cheese, several different strains of the dominant bacteria were present, as determined by pulsed-field gel electrophoresis, and many of the less common bacteria were present as single clones. The culture-independent method, denaturing gradient gel electrophoresis, resulted in identification of several bacteria which were not found by the culture-dependent (isolation and rep-PCR identification) method. It was thus a useful complementary technique to identify other bacteria in the cheeses. The gross composition, the rate of increase in pH, and the indices of proteolysis were different in most of the cheeses.

Bioengineering of nisin to enhance functionality against dairy pathogens

The bacteriocin class of antimicrobial peptides have emerged as a viable alternative to at least partially fill the void created by the end of the golden age of antibiotic discovery. Along with this potential use in a clinical setting, bacteriocins also play an important role as bio-preservatives in the food industry. This thesis focuses on a specific bacteriocin group, the lantibiotics (Lanthionine-containing antibiotics). Their numerous methods of appliance in a food setting and how their gene-encoded nature can be modified to improve on overall bioactivity and functionality are explored here. The use of a lantibiotic (lacticin 3147) producing starter culture to control the Crohn’s disease-linked pathogen Mycobacterium paratuberculosis was assessed in a raw milk cheese. Although lacticin 3147 production did not effectively control the pathogen, the study provided an impetus to employ a variety of PCR-based mutagenesis techniques with a view to the creation of enhanced lantibiotic derivatives. Through the use of these techniques, a number of enhanced derivatives were generated from the ‘hinge’ region of the nisin peptide. Furthermore, a derivative in which the three hinge amino acids were replaced with three alanines represents the first enhanced derivative of nisin to have been designed through a rational process. This derivative also formed the backbone for the creation of an active, trypsin resistant, variant. Through the employment of further mutagenesis methods a derivative was created with potential use as an oral anti-bacterial in the future. Finally a number of lead nisin derivatives were investigated to assess their anti- Streptococcus agalactiae ability, a mastitis associated pathogen. Also a system was developed to facilitate the large scale production of these candidates, or other nisin derivatives, from dairy substrates.

Comparative genome analysis of human bifidobacteria with commercial potential

This thesis describes two newly sequenced B. longum subsp. longum genomes and subsequent comparative analysis with publicly available B. longum subsp. longum, B. longum subsp. infantis and B. longum subsp. suis genomes (Chapter 2). The acquired data revealed a closed pan-genome for this bifidobacterial species and furthermore facilitated the definition of the B. longum core genome. The comparative analysis also highlights differences in the potential metabolic abilities of all three sub-species. Interestingly, phylogenetic analysis of the B. longum core genome indicated the existence of a novel B. longum subspecies. Characterisation of restriction-modification systems from two B. longum subsp. longum strains is described in Chapter 3. These defence mechanisms limit the uptake of genetic material, which was successfully demonstrated for some of the identified systems. When these systems were by-passed by methylation of DNA prior to the transformation procedure, the resulting transformation efficiency of both B. longum subsp. longum strains was increased to a level that allowed for the generation of mutants via homologous recombination. Arabinoxylan metabolism by B. longum subsp. longum NCIMB 8809 was investigated in Chapter 4 of this thesis. Transcriptome analysis allowed the identification of a number of genes involved in the degradation, uptake and utilisation of arabinoxylan. Biochemical analysis revealed that three of the identified genes encode arabinofuranosidase activity. Phenotypic assessment of a number of insertion mutants in genes identified by the transcriptome analysis revealed the essential role of two of these enzymes in arabinoxylan metabolism, and a third enzyme in the metabolism of debranched arabinan. Furthermore, this investigation revealed that B. longum subsp. longum NCIMB 8809 does not completely degrade arabinoxylan, but utilises the arabinose substitutions only, while leaving the xylan backbone untouched.Finally, Chapter 5 outlines that B. longum subsp. longum NCIMB 8809 is capable of removing ferulic and p-coumaric acid substitutions that originate from arabinoxylan. Analysis of the genome sequence led to the identification of a candidate gene for this activity, which was subsequently cloned and expressed in E. coli. Biochemical analysis revealed that the enzyme, designated here as FaeA, is indeed capable of releasing both ferulic and p-coumaric acid from arabinoxylan. Furthermore, it is shown that a derivative of B. longum subsp. longum NCIMB 8809 carrying an insertion mutation in faeA had lost the ability to release ferulic and p-coumaric acid from arabinoxylan, and that growth of this mutant strain is negatively affected when cultivated on growth-limiting levels of arabinoxylan.

Valutazione dell'attivita prebiotica di equiseto e residui della lavorazione degli agrumi

Obiettivo di questa sperimentazione è stata la valutazione in vitro dell’attività prebiotica di un sottoprodotto dell’industria alimentare, il pastazzo di agrumi, ed una pianta officinale largamente diffusa in natura, l’equiseto, nei confronti di alcuni batteri lattici isolati da feci di origine umana. Come riferimento si sono utilizzati due composti a riconosciuta attività prebiotica, l’inulina ed i frutto-oligosaccaridi (FOS), e ceppi di Bifidobacterium isolati da un preparato commerciale (Bifiselle®, Bromatech). I ceppi batterici utilizzati per tale prova sono stati isolati da campioni fecali di individui caratterizzati da differenti regimi alimentari – onnivoro, vegano od ovo-latto vegetariano -nell’ambito delle attività relative al progetto PRIN 2010-2011 “Microrganismi negli alimenti e nell'uomo: studio del microbiota e del relativo metaboloma in funzione della dieta onnivora, vegetariana o vegana (Gut4Diet)”. I risultati ottenuti hanno messo in evidenza come alcuni dei ceppi studiati posseggano una buona capacità di crescita in terreni di coltura con sottoprodotti agrumari ed equiseto come fonti di carbonio. Tramite analisi delle molecole volatili si sono determinate più di 60 molecole appartenenti principalmente alle classi degli acidi organici e loro esteri, alcoli, aldeidi, chetoni e pirazine. Tra queste vi sono alcuni esteri di acidi grassi a corta catena che si sono accumulati soprattutto nei sistemi addizionati di equiseto, FOS ed inulina. E’ noto che gli acidi grassi a corta catena sono prodotti dalla flora batterica intestinale durante la fermentazione di polisaccaridi non digeribili ed esplicano un ruolo protettivo nei confronti di differenti patologie.

Effect of galactose metabolising and non-metabolising strains of Streptococcus thermophilus as a starter culture adjunct on the properties of Cheddar cheese made with low or high pH at whey drainage

Cheddar cheese was made using control culture (Lactococcus lactis subsp. lactis), or with control culture plus a galactose-metabolising (Gal+) or galactose-non-metabolising (Gal-) Streptococcus thermophilus adjunct; for each culture type, the pH at whey drainage was either low (pH 6.15) or high (pH 6.45). Sc. thermophilus affected the levels of residual lactose and galactose, and the volatile compound profile and sensory properties of the mature cheese (270 d) to an extent dependent on the drain pH and phenotype (Gal+ or Gal-). For all culture systems, reducing drain pH resulted in lower levels of moisture and lactic acid, a higher concentration of free amino acids, and higher firmness. The results indicate that Sc. thermophilus may be used to diversify the sensory properties of Cheddar cheese, for example from a fruity buttery odour and creamy flavour to a more acid taste, rancid odour, and a sweaty cheese flavour at high drain pH.

Produção de bioetanol, usando soro do queijo como fonte de carbono. Screening de uma estirpe de levedura competente

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Produção de bioetanol por uma cultura mista de leveduras a partir de um substrato misto de alfarroba e soro de queijo

Dissertação de Mestrado, Engenharia Biológica, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2014

Selective enumeration of dairy based strains of probiotic and lactic acid bacteria

Reliable methods for selective enumeration of probiotic and lactic acid bacteria (LAB) are required for improving the functional food quality of probiotics. Various methods were evaluated for selective enumeration of seventeen LAB and probiotic strains. Tested sugars failed to select any species however, improved recovery of total LAB count. The strains were viable and physiologically active within a range of oxygen tension levels, temperature and acidic conditions. Prior methods showed varied results such as De Man Rogosa Sharpe containing bile (MRSB), MRS containing nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL), M17 and L. casei (LC) agar failed to select Lactobacillus acidophilus, Bifidobacterium, starter LAB and L. casei strains respectively. However, LC agar appears appropriate for L. paracasei and MRSB for yoghurt starter bacteria in the absence of L. reuteri and L. rhamnosus. The study suggests selective potential of culture media largely depends on target species.

Evaluation of cross-linked enzyme aggregates of Lactobacillus cell-envelope proteinases, for protein degradation

Enzymatic hydrolysis is a widely used approach to improve the functional, nutritionaland physiological properties of food proteins. In this study, cross-linked enzyme aggre-gates (CLEAs) have been prepared from cell-envelope proteinases (CEPs) of Lactobacillusdelbrueckii subsp. lactis 313 and their proteolytic properties have been evaluated using severalfood proteins. We have optimized cross-linking conditions including ammonium sulphateconcentration, incubation temperatures, agitation speed, glutaraldehyde cross-linker con-centration, reaction time and the addition of proteic feeders. Particularly, the presence ofBSA improves retained activity of cross-linked CEP aggregates (CLCEPAs) from 21.5% to 40.9%.Blocking unreacted cross-linking groups on aggregates is important to enhance recyclabil-ity of CLCEPAs. CLCEPAs had attractive thermal stability at 50◦C and it showed enhancedcatalytic activity over long-term storage after lyophilization. We have demonstrated thatCLCEPAs has proteolytic properties on different food proteins including complex (chickenegg albumin, skimmed-milk protein), fractionated (bovine casein, whey protein isolate), andpurified (bovine serum albumin) proteins. Being the first report of CLEAs from lactobacilliCEPs, this study demonstrates the feasibility of using LDL 313 CLCEPAs for degradation ofvarious food proteins.

Survival and fermentation activity of probiotic bacteria and oxidative stability of omega-3 oil in co-microcapsules during storage

Tuna oil (O) and probiotic bacteria Lactobacillus casei (P) were co-microencapsulated in whey protein isolate (WPI)-gum Arabic (GA) complex coacervate. The co-microcapsules (WPI-P-O-GA), L. casei microcapsules (WPI-P-GA) and tuna oil microcapsules (WPI-O-GA) were converted into powder using spray and freeze drying. The interaction between probiotic bacteria and omega-3 oil in co-microcapsules, particularly in terms of oxidative stability of omega-3 oil and vitality/viability of probiotic bacteria and any synergistic outcome, was studied. The effect of storage temperature (5 and 25 °C) and time (90 days) on the survival and fermentation activity of L. casei and oxidative stability of tuna oil in the microcapsules/co-microcapsules was determined. A synergism between oxidative stability of omega-3 oil and vitality of probiotic bacteria was observed, when they were co-microencapsulated and spray dried. These co-microcapsules will likely have utility in functional food formulations due to simple and cost effective stabilisation and delivery of two important functional ingredients.

Analysis of the genome content of Lactococcus garvieae by genomic interspecies microarray hybridization

BACKGROUND Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study, the genomic content of L. garvieae CECT 4531 was analysed using bioinformatics tools and microarray-based comparative genomic hybridization (CGH) experiments. Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 were used as reference microorganisms. RESULTS The combination and integration of in silico analyses and in vitro CGH experiments, performed in comparison with the reference microorganisms, allowed establishment of an inter-species hybridization framework with a detection threshold based on a sequence similarity of >or= 70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of the genes are related to ribosomal, sugar metabolism or energy conversion systems. Some of the identified genes, such as als and mycA, could be involved in the pathogenesis of L. garvieae infections. CONCLUSIONS In this study, we identified 267 genes that were potentially present in L. garvieae CECT 4531. Some of the identified genes could be involved in the pathogenesis of L. garvieae infections. These results provide the first insight into the genome content of L. garvieae.