Application of Fermentation Techniques in the Utilization of Prawn Shell Waste

The present study aimed at the utlisation of microbial organisms for the production of good quality chitin and chitosan. The three strains used for the study were Lactobacillus plantarum, Lactobacililus brevis and Bacillus subtilis. These strains were selected on the basis of their acid producing ability to reduce the pH of the fermenting substrates to prevent spoilage and thus caused demineralisation of the shell. Besides, the proteolytic enzymes in these strains acted on proteinaceous covering of shrimp and thus caused deprotenisation of shrimp shell waste. Thus the two processes involved in chitin production can be affected to certain extent using bacterial fermentation of shrimp shell.Optimization parameters like fermentation period, quantity of inoculum, type of sugar, concentration of sugar etc. for fermentation with three different strains were studied. For these, parameters like pH, Total titrable acidity (TTA), changes in sugar concentration, changes in microbial count, sensory changes etc. were studied.Fermentation study with Lactobacillus plantarum was continued with 20% w/v jaggery broth for 15 days. The inoculum prepared yislded a cell concentration of approximately 108 CFU/ml. In the present study, lactic acid and dilute hydrochloric acid were used for initial pH adjustment because; without adjusting the initial pH, it took more than 5 hours for the lactic acid bacteria to convert glucose to lactic acid and during this delay spoilage occurred due to putrefying enzymes active at neutral or higher pH. During the fermentation study, pH first decreased in correspondence with increase in TTA values. This showed a clear indication of acid production by the strain. This trend continued till their proteolytic activity showed an increasing trend. When the available sugar source started depleting, proteolytic activity also decreased and pH increased. This was clearly reflected in the sensory evaluation results. Lactic acid treated samples showed greater extent of demineralization and deprotenisation at the end of fermentation study than hydrochloric acid treated samples. It can be due to the effect of strong hydrochloric acid on the initial microbial count, which directly affects the fermentation process. At the end of fermentation, about 76.5% of ash was removed in lactic acid treated samples and 71.8% in hydrochloric acid treated samples; 72.8% of proteins in lactic acid treated samples and 70.6% in hydrochloric acid treated samples.The residual protein and ash in the fermented residue were reduced to permissible limit by treatment with 0.8N HCI and 1M NaOH. Characteristics of chitin like chitin content, ash content, protein content, % of N- acetylation etc. were studied. Quality characteristics like viscosity, degree of deacetylation and molecular weight of chitosan prepared were also compared. The chitosan samples prepared from lactic acid treated showed high viscosity than HCI treated samples. But degree of deacetylation is more in HCI treated samples than lactic acid treated ones. Characteristics of protein liquor obtained like its biogenic composition, amino acid composition, total volatile base nitrogen, alpha amino nitrogen etc. also were studied to find out its suitability as animal feed supplement.Optimization of fermentation parameters for Lactobacillus brevis fermentation study was also conducted and parameters were standardized. Then detailed fermentation study was done in 20%wlv jaggery broth for 17 days. Also the effect of two different acid treatments (mild HCI and lactic acid) used for initial pH adjustment on chitin production were also studied. In this study also trend of changes in pH. changes in sugar concentration ,microbial count changes were similar to Lactobacillus plantarum studies. At the end of fermentation, residual protein in the samples were only 32.48% in HCI treated samples and 31.85% in lactic acid treated samples. The residual ash content was about 33.68% in HCI treated ones and 32.52% in lactic acid treated ones. The fermented residue was converted to chitin with good characteristics by treatment with 1.2MNaOH and 1NHCI.Characteristics of chitin samples prepared were studied and extent of Nacetylation was about 84% in HCI treated chitin and 85%in lactic acid treated ones assessed from FTIR spectrum. Chitosan was prepared from these samples by usual chemical method and its extent of solubility, degree of deacetylation, viscosity and molecular weight etc were studied. The values of viscosity and molecular weight of the samples prepared were comparatively less than the chitosan prepared by Lactobacillus plantarum fermentation. Characteristics of protein liquor obtained were analyzed to determine its quality and is suitability as animal feed supplement.Another strain used for the study was Bacillus subtilis and fermentation was carried out in 20%w/v jaggery broth for 15 days. It was found that Bacillus subtilis was more efficient than other Lactobacillus species for deprotenisation and demineralization. This was mainly due to the difference in the proteolytic nature of the strains. About 84% of protein and 72% of ash were removed at the end of fermentation. Considering the statistical significance (P

Studies on exopolysaccharide production by probiotic Lactic acid bacteria

The thesis mainly discussed the isolation and identification of a probiotic Lactobacillus plantarum, fermentative production of exopolysaccharide by the strain, its purification, structural characterisation and possible applications in food industry and therapeutics. The studies on the probiotic characterization explored the tolerance of the isolated LAB cultures to acid, bile, phenol, salt and mucin binding. These are some of the key factors that could satisfy the criteria for probiotic strains . The important factors required for a high EPS production in submerged fermentation was investigated with a collection of statistical and mathematical approach. Chapter 5 of the thesis explains the structural elucidation of EPS employing spectroscopic and chromatographic techniques. The studies helped in the exploration of the hetero-polysaccharide sequence from L. plantarum MTCC 9510. The thesis also explored the bioactivities of EPS from L. plantarum. As majority of chemical compounds identified as anti-cancerous are toxic to normal cells, the discovery and identification of new safe drugs has become an important goal of research in the biomedical sciences. The thesis has explored the anti-oxidant, anti-tumour and immunomodulating properties of EPS purified from Lactobacillus plantarum. The presence of (1, 3) linkages and its molecular weight presented the EPS with anti-oxidant, anti-tumour and immunomodulating properties under in vitro conditions.

Diversity and antimicrobial activity of Lactic Acid Bacteria from the gut of marine fish Rastrelliger kanagurta against fish, shrimp and human pathogens

Emergence of antibiotic resistance among aquaculture pathogens has made it necessary to look into environment friendly, effective and sustainable methods such as probiotic and immunostimulants among others.. In the present study, LAB were isolated from the gut of fish species namely Rastrelliger kanagurta and analyzed for their antibacterial activity against various fish, shrimp and human pathogens. Different LAB species such as Lactobacillus plantarum, L. bulgaricus, L. brevis and L. viridiscens were encountered in the gut of R. kanagurta. Several strains showed good activity against fish, shrimp and human pathogens. LAB from the gut of such marine species may be developed as possible probiont for environment friendly health management of fresh water, estuarine and marine species currently exploited in aquaculture

Gut Associated Lactic Acid Bacteria Isolated from the Estuarine Fish Mugil cephalus: Molecular Diversity and Antibacterial Activities against Pathogens

In the present study we address the issue on gut associated lactic acid bacteria (LAB) isolated from the intestine of estuarine fish Mugil cephalus using de Man Rogossa and Sharpe (MRS) agar. LAB isolates were identified biochemically and screened for their ability to inhibit in vitro growth of various fish, shrimp and human pathogens. Most of the LAB isolates displayed an improved antagonism against fish pathogens compared to shrimp and human pathogens. Selected representative strains displaying high antibacterial activity were identified using 16S rRNA gene sequence analysis. Of the selected strains Lactobacillus brevis was the most predominant. Four other species of Lactobacillus, Enterobacter hormaechei and Enterobacter ludwigii were also identified. It was also observed that even among same species, considerable diversity with respect to substrate utilization persisted. Considering the euryhaline nature of grey mullet (Mugil cephalus), the LAB isolated from the gut possessed good tolerance to varying salt concentrations. This finding merits further investigation to evaluate whether the isolated LAB could be used as probiotics in various fresh and sea water aquaculture

Probiotic Effects of Lactic Acid Bacteria Against Vibrio Alginolyticus in Penaeus (Fenneropenaeus) Indicus (H. Milne Edwards)

Cell free extracts of four strains of Lactic acid bacteria (LAB) viz. Lactobacillus. acidophilus, Streptococcus.cremoris, Lactobacillus bulgaricus –56 and Lactobacillus bulgaricus –57 inhibited growth of Vibrio alginolyticus in nutrient broth. The antagonism of LAB to Vibrio alginolyticus was further confirmed by streak plating wherein suppression of growth of Vibrio was obtained. Juveniles of Penaeus indicus (average weight 0.985 ± 0.1 g) on administering orally a moist feed base containing 5 × 106 cells·g of the four LAB probionts for a period of four weeks showed better survival (56 to 72%) when challenged with V. alginolyticus by intra-muscular injection of 0.1 ml containing 3 × 109 cells·ml. Animals maintained on a diet devoid of bacterial biomass exhibited 80% mortality. No external or internal pathological changes were observed in shrimp fed with the LAB incorporated diets. Results showed inhibition of V. alginolyticus by LAB and stimulation of the non-specific immune response resulting in resistance to disease in the shrimp fed on LAB incorporated diets.

Untersuchungen über die Redoxregulation der Glutathionsynthese und Charakterisierung von Glutaredoxinen in Spinat

Die an der Glutathionsynthese im Chloroplasten von Spinatblättern beteiligten Enzyme sind auf eine lichtabhängige Regulation durch Thioredoxine (Trx) und Glutaredoxine (Grx) hin untersucht worden. Dazu wurde eine neue, vereinfachte Methode zur Aktivitätsbestimmung für die gamma-Glutamylcystein- und Glutathionsynthetase auf der Kapillarelektrophorese entwickelt. Untersuchungen mit den homologen Thioredoxinen Trx m und Trx f aus Spinatchloroplasten und mit dem E.coli Trx und E.coli Grx 1 zeigten, dass bei beiden Enzymen keine Redoxmodulation durch diese Proteine stattfindet. Weitere Untersuchungen mit der Glutathionsynthetase zeigten keinen Einfluss von Dithiothreit, Sulfit-Ionen und Ascorbat auf die Enzymaktivität. Nur H2O2, in unphysiologischen Konzentrationen, bewirkte eine leichte Abnahme der Ausgangsaktivität. Im Fall der gamma-Glutamylcysteinsynthetase konnten verschiedene Einflüsse ausgemacht werden. So war mit Dithiothreit und H2O2 bei niedrigen Konzentrationen eine Stimulation und bei höheren Konzentration eine Inhibition der Enzymaktivität festzustellen: Sulfit-Ionen zeigten eine starke Stimulierung der gamma-Glutamylcysteinsynthetase über einen weiten Konzentrationsbereich, wobei eine starke pH-Wert-Abhängigkeit der Stimulation zu beobachten war. Ascorbat zeigte, wie bei der Glutathionsynthetase, keinen Einfluss auf die Enzymaktivität der gamma-Glutamyl-cysteinsynthetase. In einem zweiten Teil der Arbeit über die Glutaredoxine des Spinats konnte ein 12,4 kDa Protein mit Thioltransferase-Aktivität, das bisher als cytosolisches Glutaredoxin beschrieben wurde, aufgereinigt und mittels N-terminaler Sequenzierung eindeutig als ein Glutaredoxin identifiziert werden. Überdies konnte ein noch nicht beschriebenes 12,8 kDa Protein mit Thioltransferase-Aktivität aus Spinatchloroplasten aufgereinigt werden. Durch Peptid-Sequenzierung gelang es dieses Protein auch als ein Glutaredoxin zu identifizieren. Beide pflanzlichen Glutaredoxine zeigten keine Modulation der Aktivitäten der chloroplastidären Fructosebisphosphatase (FbPase) und NADPH-Malatdehydrogenase (NADPH-MDH). Auch war mit beiden Glutaredoxinen keine Dehydroascorbatreduktase-Aktivität, oder eine Stimulation der Ribonucleotidreduktase aus Lactobacillus leichmannii festzustellen.

Estudio de las comunidades microbianas de embutidos fermentados ligeramente acidificados mediante técnicas moleculares. Estandarización, seguridad y mejora tecnológica.

Los embutidos fermentados ligeramente acidificados son un grupo de productos tradicionales mediterráneos, caracterizados por un pH superior a 5,3. Para un control eficiente de la seguridad microbiológica de los embutidos se necesitan técnicas rápidas para la identificación y recuento de los microorganismos patógenos a estudiar. En el presente trabajo, se desarrolló una técnica para la enumeración de L. monocytogenes que combinó el método del número más probable y la identificación mediante PCR específica. Para la detección de Salmonella spp. y L. monocytogenes se desarrolló un sistema de PCR-multiplex que permitió la identificación de ambos patógenos de forma simultánea en una sola reacción. El estudio de la calidad microbiológica de los embutidos fermentados ligeramente acidificados se completó con la caracterización de las comunidades microbianas más importantes en estos productos. Se identificaron a nivel de especie los aislados de bacterias del ácido láctico (BAL), de enterococos y de cocos gram-positivos catalasa-positivos (CGC+). Posteriormente se realizó una tipificación molecular de los mismos mediante RAPD y análisis del perfil plasmídico y se estudiaron las principales características de interés higiénico-sanitario y tecnológico de las cepas. Mediante PCR se identificó Lactobacillus sakei como la especie predominante (74%), seguida por Lactobacillus curvatus (21,2%). La actividad aminoácido-descarboxilasa se asoció a la especie L. curvatus (el 66% de los aislados presentaron esta actividad). La identificación de los enterococos se realizó mediante PCR-multiplex y por secuenciación del gen sodA. Enterococcus faecium fue la especie de enterococos predominante (51,9%) seguida por Enterococcus faecalis (14,2%). Todas las cepas de E. faecalis presentaron genes asociados a factores de virulencia. E. faecalis presentó mayor resistencia a antibióticos que el resto de las especies de enterococos estudiadas. Tan sólo una cepa de E. faecium presentó el genotipo vanA (que confiere resistencia de alto nivel a la vancomicina). La identificación de los aislados de CGC+ (mediante PCR específica y amplificación de la región intergénica 16S-23S ARNr) demostró que Staphylococcus xylosus es la especie predominante en los embutidos fermentados ligeramente acidificados (80,8%). La amina biógena más común en los CGC+ fue la feniletilamina, producida por un 10,8% de aislados. Un pequeño porcentaje de aislados fueron mecA+ (4,6%), presentando además resistencia a múltiples antibióticos. El potencial enterotoxigénico de las cepas de CGC+ fue muy reducido (3,3% de los aislados), detectándose únicamente el gen entC. El estudio pormenorizado de las comunidades bacterianas de interés permitió la selección de 2 cepas de L. sakei y 2 cepas de S. xylosus con características tecnológicas e higiénico-sanitarias óptimas. Para evaluar su efectividad como cultivos iniciadores se elaboraron dos tipos de embutidos ligeramente ácidos, chorizo y fuet, inoculados con microorganismos patógenos (Salmonella spp., L. monocytogenes y S. aureus). El uso de cultivos iniciadores permitió el control de L. monocytogenes, Enterobacteriaceae y Enterococcus así como del contenido en aminas biógenas. Los recuentos de Salmonella spp. disminuyeron de forma significante durante la maduración de los embutidos, independientemente del uso de cultivos iniciadores. El uso del tratamiento de alta presión (400 MPa) en los embutidos madurados consiguió la ausencia de Salmonella spp. en los lotes tratados.

Caracterització de Lactobacils d'origen intestinal i avaluació in vivo del seu poder probiòtic en pollastres

Una soca de Lactobacillus salivarius resistent a la rifampicina, CTC2197, es va assajar com a probiòtic en pollastres, estudiant la seva capacitat de prevenir la colonització de Salmonella enteritidis C-114 en pollastres. Quan la soca probiòtica es va administrar via oral juntament amb S.enteritidis C-114 directament al proventricle en pollets Leghorn de 1 dia, el patògen fou eliminat completament després de 21 dies. Els mateixos resultats es van obtenir quan la soca es va administrar a través del menjar i l'aigua a més de la inoculació directa al proventricle. La inclusió de L.salivarius CTC2197 en el menjar del primer dia va mostrar que una concentració de 105 UFC g-1 era suficient per assegurar la colonització dels tracte gastrointestinal dels pollets després de 1 setmana. No obstant, entre els 21 i 28 dies, L.salivarius CTC2197 no va ser detectable en el tracte gastrointestinal d'alguns pollets, mostrant que seria necessària més d'una dosis per assegurar la seva presència fins al final de l'etapa d'engreix. La liofilització i la congelació per glicerol o llet descremada com a agents crioprotector, van semblar mètodes adequats per preservar la soca probiòtica. La inclusió de L.salivarius CTC2197 en un pinso comercial va semblar ser un bon mètode per subministrar-lo en granja, tot i que la soca va mostrar sensibilitat a les temperatures utilitzades durant l'emmagatzematge del pinso i a les incubadores dels pollets. A més, la supervivència va millorar després de diverses reinoculacions en pinso.

Aislamiento, identificación y conservación de cultivos de bacterias lácticas antagonistas de microbiota contaminante de sangre de matadero

La sangre obtenida en el matadero es un producto altamente contaminado que requiere un procesamiento inmediato si se pretende utilizarla como insumo alimentario en la fabricación de productos destinados al consumo humano. Si bien es cierto que los sistemas de higienización podrían ser muy eficientes desde el punto de vista de calidad microbiológica, su instalación en la línea de sacrificio comportaría muchas dificultades desde el punto de vista técnico y en algún caso sería muy costoso. La bioconservación podría ser una alternativa para mejorar la calidad microbiológica de la sangre, alargar su vida útil y reducir las posibilidades de procesamiento inmediato. El presente estudio nos permitiría formular la posibilidad de aplicar la bioconservación en sangre de cerdo procedente de matadero industrial, utilizando bacterias ácido lácticas (BAL) como cultivo bioprotector, para lo cual se aislaron cepas de BAL autóctonas y se confeccionaron dos colecciones una de BAL mesófilas y otra de psicrótrofas. Se evaluó el potencial antagonista de la colección de BAL mesófilas y psicrótrofas a 30ºC y a 15ºC respectivamente frente a bacterias contaminantes habituales de este subproducto. Las BAL que demostraron antagonismo en placa (7,1% a 30ºC y 11% a 15ºC) fueron seleccionadas para evaluar el potencial antagonista en sangre, donde el efecto inhibitorio se vio favorecido por la adición de un 2% glucosa. S.aureus y P. fluorescens fueron los indicadores más inhibidos por las cepas mesófilas, en algunos casos con reducciones superiores a 7 unidades logarítmicas. En condiciones psicrótrofas la bacteria más sensible a la presencia de BAL fue Bacillus sp., donde 8 de las 11 BAL ensayadas permitieron reducciones superiores a 4 logs y 1cepa incluso superiores a 7 logs; se obtuvieron reducciones máximas de 3 logs de E.coli y Pseudomonas fue inhibida por todas las BAL ensayadas, en algún caso con reducciones superiores a 5 logs. Las 5 que cepas que presentaron el espectro de inhibición más amplio en condiciones mesófilas y 7 en condiciones psicrótrofas frente a los microorganismos indicadores contaminantes de sangre de matadero se identificaron mediante técnicas moleculares por comparación de la secuencias correspondientes al gen que codifica la síntesis de 16S ARNr (16S ADNr) con las secuencias publicadas en las bases de datos. De las 7 cepas antagonistas en condiciones psicrótrofas 5 se identificaron como Lactococcus garvieae y 2 como Enterococcus malodoratus/gilvius raffinosus. Todas las BAL con potencial antagonista en condiciones mesófilas pertenecían al género Lactobacillus, 3 de elllas se identificaron como Lactobacillus murinus/animalis y una se identificó como Lactobacillus reuteri. TA20 que tuvo un gran espectro de inhibición a ambas temperaturas se identificó como Lactococcus garvieae. En este estudio se evaluaron tres métodos de conservación a largo plazo de las cepas que mostraron potencial antagonista. Se comparó la liofilización, la atomización frente a la congelación a -80ºC que era método que se había utilizado hasta el momento para conservar ambas colecciones de BAL. En general, los métodos de deshidratación (atomización y liofilización) y mantenimiento en refrigeración a 5ºC de los cultivos deshidratados se han mostrado más eficaces que la congelación.

Mixed culture fermentation studies on the effects of synbiotics on the human intestinal pathogens Campylobacter jejuni and Escherichia coli

Batch and continuous culture anaerobic fermentation systems, inoculated with human faeces, were utilised to investigate the antimicrobial actions of two probiotics, Lactobacillus plantartan 0407, combined with oligofructose and Bifidobacterium bifidum Bb12, combined with a mixture of oligofructose and xylo-oligosaccharides (50:50 w/w) against E coli and Campylobacter jejuni. In batch fermenters, both E coli and C jejuni were inhibited by the synbiotics, even when the culture pH was maintained at around neutral. In continuous culture C jejuni was inhibited but the synbiotic failed to inhibit E coli. Although no definitive answer in addressing the mechanisms underlying antimicrobial activity was derived, results suggested that acetate and lactate directly were conferring antagonistic action, rather than as a result of lowering culture pH. In the course of the study culturing and fluorescent in situ hybridisation (FISH) methodologies for the enumeration of bacterial populations were compared. Bifidobacterial populations were underestimated using plating techniques, suggesting the non-culturability of certain bifidobacterial species. (C) 2003 Elsevier Ltd. All rights reserved.

In vitro three-stage continuous fermentation of gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 by the human colonic microflora

Gluco-oligosaccharides produced by Gluconobacter oxydans NCIMB 4943 from maltodextrin as the source, were evaluated for their fermentability by the human colonic microflora. The selectivity of growth of desirable bacteria in the human colon was studied in a three-stage continuous model of the human large intestine. Populations of bacteria, and their fluctuations as a response to the fermentation, were enumerated using fluorescent in situ hybridization (FISH). The gluco-oligosaccharides resulted in increases in numbers of bifidobacteria and the Lactobacillus/Enterococcus group in all 3 vessels of the system, representing the proximal, transverse and distal colonic areas. The prebiotic indices of the glucooligosaccharides were 2.29, 4.23 and 2.74 in V1, V2 and V3 respectively.

Developing a quantitative approach for determining the in vitro prebiotic potential of dietary oligosaccharides

Prebiotics are nondigestible carbohydrates that beneficially affect the host by selectively stimulating the growth and/or activity of one, or a limited number of, bacteria present in the colon. The selected genera should have the capacity to improve host health (e.g. Bifidobacterium, Lactobacillus). To help identify preferred types, for inclusion into the diet, a quantitative equation [measure of the prebiotic effect (MPE)] is suggested. This will help evaluate, in vitro, the fermentation of dietary carbohydrates and compare their prebiotic effect. Although the approach is not meant to define health values, it is formulated to better inform the choice of prebiotic. It therefore, compares measurements of bacterial changes through the determination of maximum growth rates of predominant groups present in faeces, rate of substrate assimilation and the production of lactic, acetic, propionic and butyric acids. The equation will allow further in vitro comparisons of MPE, leading towards further studies (e.g. in humans) to determine the success of dietary intervention. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Biodiversity of human faecal bacteria isolated from phytic acid enriched chemostat fermenters

Background: Myo-inositol hexaphosphate (IP6) or phytic acid is found mostly in cereals and legumes and is thought to possess anti-carcinogenic properties. Aim: To isolate and identify faecal bacteria capable of phytic acid metabolism and to assess the effectiveness of prebiotics (dietary oligosaccharides, metabolised by selective colonic bacteria) in preserving the integrity of phytic acid. Methods: Faecal samples from three volunteers were used in continuous culture experiments under varying conditions of pH, substrate concentration and dilution rates, seventy three different isolates cultured at steady state were then screened for phytic acid metabolism and identified through partial sequencing of their 16S rRNA genes (16S ribosomal ribonucleic acid). Utilisation of phytic acid was also assessed in a continuous culture system enriched with prebiotic fructooligosaccharides (FOS). Results: Bacteroides spp., Clostridium spp. and facultatively anaerobic bacteria generally appeared to maintain viable counts in the presence of phytic acid. Bifidobacterium spp. and Lactobacillus spp. appeared less able to maintain viable counts in the presence of phytic acid. These results were confirmed by an increase in viable counts of Bacteroides spp., Clostridium spp. and a decrease in viable counts of Bifidobacterium spp. and Lactobacillus spp. once phytic acid was introduced to a FOS enriched continuous culture. Conclusions: The phytate metabolising biodiversity from the human large intestine does not appear to encompass major bacterial genera associated with beneficial or benign health effects (e.g. Lactobacillus spp. and Bifidobacterium spp).

Metabolism of the soyabean isoflavone glycoside genistin in vitro by human gut bacteria and the effect of prebiotics

The isoflavone genistein is found predominantly in soyabeans and is thought to possess various potent biological properties, including anticarcinogenic effects. Studies have shown that genistein is extensively degraded by the human gut microflora, presumably with a loss of its anti-carcinogenic action. The aim of the present study was to investigate the potential of a prebiotic to divert bacterial metabolism away from genistein breakdown: this may be of benefit to the host. Faecal samples were obtained from healthy volunteers and fermented in the presence of a source of soyabean isoflavones (Novasoy(TM) (10 g/l); ADM Neutraceuticals, Erith, Kent, UK). Bacterial genera of the human gut were enumerated using selective agars and genistein was quantified by HPLC. The experiment was repeated with the addition of glucose (10 g/l) or fructo-oligosaccharide (10 g/l; FOS) to the fermentation medium. The results showed most notably that counts of Bifidobacterium spp. and Lactobacillus spp. were significantly increased (P<0.05 and P<0.01 respectively) under steady-state conditions in the presence of FOS. Counts of Bacteroides spp. and Clostridium spp. were, however, both significantly reduced (P<0.05) during the fermentation. A decline in genistein concentration by about 52 and 56% over the 120h culture period was observed with the addition of glucose or FOS to the basal medium (P<0.01), compared with about 91% loss of genistein in the vessels containing Novasoy(TM) (ADM Neutraceuticals) only. Similar trends were obtained using a three-stage chemostat (gut model), in which once again the degradation of genistein was about 22% in vessel one, about 24% in vessel two and about 26% in vessel three in the presence of FOS, compared with a degradation of genistein of about 67% in vessel one, about 95% in vessel two and about 93% in vessel three in the gut model containing Novasoy(TM) (ADM Neutraceuticals) only. The present study has shown that the addition of excess substrate appeared to preserve genistein in vitro. In particular, the use of FOS not only augmented this effect, but also conferred an additional benefit in selectively increasing numbers of purportedly beneficial bacteria such as bifidobacteria and lactobacilli.

Effect of culture medium and cryoprotectants on the growth and survival of probiotic lactobacilli during freeze drying

To investigate the effects of the medium and cryoprotective agents used on the growth and survival of Lactobacillus plantarum and Lactobacillus rhamnosus GG during freeze drying. A complex medium was developed consisting primarily of glucose, yeast extract and vegetable-derived peptone. Trehalose, sucrose and sorbitol were examined for their ability to protect the cells during freeze drying. Using standardized amount of cells and the optimized freeze drying media, the effect of the growth medium on cell survival during freeze drying was investigated. The results showed that glucose and yeast extract were the most important growth factors, while sucrose offered better protection than trehalose and sorbitol during freeze drying. When the cells were grown under carbon limiting conditions, their survival during freeze drying was significantly decreased. A clear relationship was observed between cell growth and the ability of the cells to survive during the freeze drying process. The survival of probiotic strains during freeze drying was shown to be dependent on the cryoprotectant used and the growth medium.