Improvement in the quality of a fermented seaweed beverage using an antiyeast starter of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> DW3 and partial sterilization

The quality of a fermented beverage (FSB) produced from seaweed (Gracilaria fisheri) was investigated after four different fermentation processes. 1, a normal fermentation as control (N-N); 2, batch with addition of an inoculum of an antiyeast starter culture, Lactobacillus plantarum DW3 (N-S); 3, a partial sterilization of the seaweed with 0.5% potassium metabisulfite (KMS) (P-N); and 4, a partial sterilization followed by an inoculum as for 2 (P-S). At the end of fermentation (60 days) and after storage for 3 months, all treatment sets passed the microbiological quality guidelines, as no bacterial indicators (total coliforms and Escherichia coli) or foodborne pathogens (Salmonella sp. Clostridium perfringens and Staphylococcus aureus) were detected. All treatments improved the availability of elements (Cu, Zn, and Fe) in the seaweed beverage and they were all below the recommended safety levels. Toxic compounds such as methanol, and the elements As and Pb were below either the detection or safety limits. The starter culture controlled yeast contamination and had inhibitory effects against foodborne pathogenic bacteria (Vibrio parahaemolyticus PSSCMI 0064 > Bacillus cereus ATCC 11778 > Salmonella typhi PSSCMI 0034 ∼ Staphylococcus aureus PSSCMI 0004 ∼ E. coli PSSCMI 0001). The inoculation set without pretreatment by KMS (N-S) was the treatment that produced the best FSB based on its antibacterial activity, reduction of contaminated yeasts, remaining probiotic LAB and organoleptic properties.     

Investigation of the effects of oxidative stress-inducing factors on culturing and productivity of Bordetella pertussis

The stress response of Bordetella pertussis during fermentation was assessed by means of fluorescence-based techniques. During the manufacturing of vaccines, B. pertussis is subjected to stress during adaptation to a new environment and operating conditions in the bioreactor, which can have harmful consequences on growth and protein yield. In this study, stress was imposed by varying the percentage of dissolved oxygen (DO) and inoculum size, and by adding rotenone and hydrogen peroxide. In this study, fluorescence spectroscopy is used as a tool for measuring oxidative stress. High levels of DO during fed-batch operation had no detrimental effect on growth, but the specific productivity of pertactin (PRN) decreased. Cultures that were started with an inoculum size that was 10 times smaller than the control resulted in significantly less PRN as compared to controls where reduction was more significant in flasks as compared to bioreactors. A comparison of filtered to heat-sterilized media revealed that filtered media offered a protective effect against H₂O₂. Heat sterilization of the media might result in the destruction of components that offer protection against oxidative stress. Nonetheless, filter sterilization on its own would be insufficient for large-scale manufacturing. It should be emphasized that the effects of these stressors while investigating for other microorganisms have not been studied for B. pertussis.     

An on-line advisory control system for the lactic acid fermentation process

A fuzzy expert system was developed for online diagnosing and controlling of bioprocesses. The system was constructed in object-oriented Smalltalk/V for diagnosing and controlling of bioprocesses. Lactic acid fermentation with an industrial strain ofLactobacillus casei was chosen as the model system. The performance of the fuzzy expert system and the knowledge base utilizing experts' knowledge and several facts obtained from the experiments were successfully validated with on-line fermentations. The fuzzy expert system could diagnose a fault on-line and give reasonable advice to the process operator. In order to achieve the diagnosing faculty, a database, a knowledge base, and both backward and forward chaining procedures were implemented employing the object-oriented programming environment. A defuzzifier was implemented in the system to achieve on-line control. In order to realize a decision-making system with a human operator and a fuzzy expert system, a new control strategy namedAdvice was also introduced. Several cultivations were carried out in order to collect knowledge on the effects concerned with inoculum properties to the process and to construct a database including standard time-course profiles. The performance of the fuzzy expert control system was successfully tested with on-line experiments.Visiting scientist from HUT at RIKEN     

Production of alcohol from sugar beet molasses without heat or filter sterilization

Among three esters of p-hydroxybenzoate, n-butyl p-hydroxybenzoate was selected as the best antimicrobial substance. Molasses medium sterilized by this ester was used as a substrate for ethanol production. n-Butyl p-hydroxybenzoate (0.15% w/v) completely inhibited the growth of free yeast cell inoculum, Ca-alginate immobilized yeast inoculum and bacterial contaminants. Immobilization of the yeast cell inoculum in Ca-alginate with castor oil (6% v/v) offered a yeast cell protection against the inhibitory effect of n-butyl p-hydroxybenzoate. The presence of castor oil in this immobilization system did not affect the metabolic activity of the yeast in beads compared to the cells immobilized without castor oil. The yeast cell beads in this system completely utilized up to 25% molasses sugar with an ethanol yield of 10.58%, equal to 83% of its theoretical value. The beads were stable and could be used successfully for seven cycles of batch fermentation. The optimum fermentation temperature using this system was 35°C. Received 21 January 1997/ Accepted in revised form 05 May 1997     

Response surface optimization of mixed substrate solid state fermentation for the production of lovastatin by Monascus purpureus

The objective of this work is to enhance the production of lovastatin using Monascus purpureus MTCC 369 in mixed substrate solid state fermentation using various solid substrates and to optimize the combination of the solid substrates by response surface methodology. Solid state fermentation was conducted in a 250 mL Erlenmeyer flask at 30°C for 14 days with initial moisture content of 40% and inoculum size of 10% active culture. Barley, long grain rice and sago starch were found to be the suitable substrates producing maximum lovastatin of 193.7 mg, 190.2 mg and 180.9 mg/g of dry solids. These substrates were further used in various combinations as designed by the central composite design for enhancing the lovastatin production using Monascus purpureus. To the best of our knowledge this is the first report on the production of lovastatin using a mixed substrate solid state fermentation using Monascus purpureus.     

Influence of Temperature and Oxygen Concentration on the Fermentation Behaviour of Candida Stellata in Mixed Fermentation with Saccharomyces Cerevisiae

Summary Candida stellata is a wine-yeast species that has been proposed for use as a starter in the wine industry in combination with selected Saccharomyces cerevisiae cultures. To improve our knowledge on the metabolic interactions between these two yeast species, we have investigated the influence of temperature and oxygen concentration on their fermentation behaviour in mixed fermentation. Trials were carried out with free and immobilized C. stellata cells followed by an inoculum of a commercial strain of S. cerevisiae, to determine the biomass evolution and the oenological profiles of the resulting wines. We show that the oxygen concentration does not influence the fermentation behaviour of the C. stellata cells, while a low temperature (16 °C) is a critical condition for the metabolic activity of C. stellata in free-cell trials. The immobilization procedures produce a significant improvement in the metabolic activity of C. stellata, with a consequent enhancement of the glycerol content also at the 16 °C fermentation temperature. High cell concentrations and the immobilization system appear to positively influence the fermentation behaviour of C. stellata. These results are of interest for the practical application of this process in winemaking.     

Effect of culture conditions on growth and sporulation ofBacillus thuringiensis subsp.israelensis and development of media for production of the protein crystal endotoxin

Summary The influence of medium composition on the inoculum and production stages of theBacillus thuringiensis subsp.israelensis bioinsecticide fermentation was investigated. Media which inhibited sporulation were selected for inoculum development stages. Bioinsecticide production media were designed to produce high cell counts and >90% sporulation in a 48h fermentation. Maximum insecticidal activity occurred at the point of maximum bacterial cell lysis/spore release. A process involving two inoculum stages and a 48h production stage in a 40 l fermenter yielded a viable cell count of 6.5 x 10⁹/ml with greater than 95% sporulation. Good correlation existed between spore counts and bioinsecticide activity.     

Demineralization of red crab shell waste by lactic acid fermentation

Lactic acid fermentation was applied to demineralize red crab shell waste using Lactobacillus paracasei subsp. tolerans KCTC-3074. Various concentrations (0, 2.5, 5.0, 10.0%) of glucose were supplemented as an initial carbon source and various amounts (2.5, 5.0, 10.0%) of the bacterial culture were inoculated as a starter. Microbial growth was very dependent on glucose concentration but little dependent on inoculum level. The pH decreased rapidly from pH 8 to pH 6 during the first day, at all three inoculum levels. At day 5 of fermentation, the 2.5, 5.0, and 10.0% inoculum levels with 10% glucose supply gave pH 5.5, 5.1, and 4.6, respectively, i.e. a decrease in pH concomitant with an increase in the inoculum level. The total titratable acidities (TTA) at day 5 for 2.5, 5.0, and 10.0% inoculum levels with 10% glucose supplement were 3.1, 4.5, and 8.3%, and the relative residual ash contents were 26.6, 25.9, and 19.0%, respectively. A negative relationship was found between pH and demineralization level (r²=0.8571), but there was a positive relationship between TTA and demineralization level (r²=0.5532).     

Development of Media and Automated Liquid Fermentation Methods to Produce Desiccation-Tolerant Propagules ofTrichoderma harzianum

A suitable medium was developed from modified Richard's medium plus V8 juice (RM8) to produce high levels of desiccation-tolerant conidia ofTrichoderma harzianumstrain 1295-22. The addition of 9% (v/v) glycerol to RM8 improved both biomass production and desiccation tolerance of the conidia ofT. harzianum.This medium was then used in a laboratory scale fermenter (1.5 liter) to determine optimal operating conditions. The optimal temperature for conidial production and desiccation tolerance improvement in the fermenter was 32°C when dissolved oxygen was maintained at 50% saturation of air, and the stirring rate was 1000 revolutions per minute. The initial water potential of the medium (with 9% glycerol) was −3.7 MPa, the pH was 6, and neither was controlled during fermentation. Changes in medium pH and dissolved oxygen were associated with the stages of morphological development and conidiation. The pH of the medium decreased concurrently with germ-tube elongation and mycelium development and then increased to 6.0–6.2 at phialide formation. Intensive conidiation occurred at pH 6.3–6.5 and reached its maximal level at 6.9–7.1. Changes in pH values could be used as indicators to monitor the morphological development and conidiation ofT. harzianumduring fermentation. The use of a 48-h-old culture inoculum, rather than conidial inoculum, to start fermentation reduced the time required to complete the shift from vegetative growth to phialide formation. Intensive conidiation occurred immediately after the addition of culture inoculum and reached maximum levels within 68 h of fermentation. Dry weight of biomass increased with the duration of fermentation and was greatest at 96 h. However, no improvements in conidia/gram and CFU/gram were achieved after 72 h of fermentation. The desiccation tolerance of conidia harvested at 72 or 96 h was significantly (P = 0.05) greater than that of conidia harvested at 48 h of fermentation. Results obtained from this study could be used for further scale-up of the fermentation process.     

Production and regulation of extracellular chitinase from the entomopathogenic fungus Isaria fumosorosea

Extracellular chitinase production by the entomopathogenic fungus, Isaria fumosorosea IF28.2 was studied by using submerged fermentation. Maximum chitinase production (178.34±3.91 mU/mL) was obtained when fermentation was carried out at 25°C for 120 h using 72-h-old mycelium in a medium. The effect of inoculum size on chitinase activity was also observed and maximum chitinase activity (159.41±2.91 mU/mL) was obtained with an inoculum size of 3 discs while an incubation period of 96 h proved the most active inducer of chitinase production yielding a chitinase activity of 186.14±3.81 mU/mL. Colloidal chitin (1.5%, w/v) proved to be the best concentration. The optimum pH for chitinase production was 5.7 while 25°C proved to be the best temperature for chitinase production. Supplementation of additional carbon source like 1.5% N-acetylglucosamine (GlcNAc) showed further enhancement in chitinase production. The divalent metal salts, CaCl₂, MgCl₂ and ZnSO₄, inhibited chitinase activity at 10 and 100 mM concentration, whereas inhibition of chitinase activity by KCl, FeSO₄ and EDTA was observed only at higher concentrations. The results presented in this study increase the knowledge on chitinase production in I. fumosoroseus opening new avenues for the study of the role of this enzyme in virulence against different insect pests during the infection process.     

Effects of high‐ and low‐fiber diets on fecal fermentation and fecal microbial populations of captive chimpanzees

We examined fiber fermentation capacity of captive chimpanzee fecal microflora from animals (n=2) eating low‐fiber diets (LFDs; 14% neutral detergent fiber (NDF) and 5% of cellulose) and high‐fiber diets (HFDs; 26% NDF and 15% of cellulose), using barley grain, meadow hay, wheat straw, and amorphous cellulose as substrates for in vitro gas production of feces. We also examined the effects of LFD or HFD on populations of eubacteria and archaea in chimpanzee feces. Fecal inoculum fermentation from the LFD animals resulted in a higher in vitro dry matter digestibility (IVDMD) and gas production than from the HFD animals. However, there was an interaction between different inocula and substrates on IVDMD, gas and methane production, and hydrogen recovery (P<0.001). On the other hand, HFD inoculum increased the production of total short‐chain fatty acids (SCFAs), acetate, and propionate with all tested substrates. The effect of the interaction between the inoculum and substrate on total SCFAs was not observed. Changes in fermentation activities were associated with changes in bacterial populations. DGGE of bacterial DNA revealed shift in population of both archaeal and eubacterial communities. However, a much more complex eubacterial population structure represented by many bands was observed compared with the less variable archaeal population in both diets. Some archaeal bands were related to the uncultured archaea from gastrointestinal tracts of homeothermic animals. Genomic DNA in the dominant eubacterial band in the HFD inoculum was confirmed to be closely related to DNA from Eubacterium biforme. Interestingly, the predominant band in the LFD inoculum represented DNA of probably new or yet‐to‐be‐sequenced species belonging to mycoplasms. Collectively, our results indicated that fecal microbial populations of the captive chimpanzees are not capable of extensive fiber fermentation; however, there was a positive effect of fiber content on SCFA production. Am. J. Primatol. 71:548–557, 2009. © 2009 Wiley‐Liss, Inc.     

Production of yeast-lytic enzymes by Cytophaga species in batch culture

The conditions for culture storage, inoculum preparation, and growth of a Cytophaga species, constitutive with respect to yeast-lytic enzymes, have been established in shake-flask studies and in 5 liter fermentor experiments. A low cost medium was adopted for 900 liter-scale fermentation and gave an enzyme activity in the fermentation broth somewhat greater than the comparable laboratory-scale one.     

Production of Fumaric Acid in 20-Liter Fermentors

The conditions necessary for the production of fumaric acid in 20-liter fermentors by fermentation of glucose with Rhizopus arrhizus strain NRRL 2582 were determined. Continuous neutralization of fumaric acid was necessary for optimal yields. Yields of the calcium salt were in excess of 65 g of fumaric acid from 100 g of sugar consumed during fermentation of sugar concentrations of 10 to 16%. Conditions established for calcium fumarate production include a simple mineral salts medium, 0.5 v:v:min aeration rate, 300 rev/min agitation rate in a baffled tank, 33 C incubation temperature, CaCO(3) to neutralize the acid formed, and a 4 to 5% (v/v) vegetative inoculum. A suitable procedure and medium for the preparation of a vigorous vegetative inoculum were established. The tendency for calcium fumarate fermentations to foam excessively was controlled with a proper antifoam agent added prior to sterilization of the medium and again at daily intervals during fermentation. The production of soluble sodium or potassium fumarates was inhibited when the concentration of fumarates reached 3.5 to 4.0%. No means of overcoming this inhibition was found. Starches and certain other grain-derived carbohydrates were fermented to form calcium fumarate in flask experiments with approximately the same efficiency as was glucose.     

Production of α-galactosidase by a novel actinomycete <Emphasis Type="Italic">Streptomyces griseoloalbus</Emphasis> and its application in soymilk hydrolysis

α-Galactosidase production by a newly isolated actinomycete Streptomyces griseoloalbus under submerged fermentation was investigated. The influence of initial pH of medium, incubation temperature, inoculum age and inoculum size on α-galactosidase formation was studied. Various carbon sources were supplemented in the medium to study their effect on enzyme production. The influence of the concentration of locust bean gum on enzyme production also was optimized. Optimization of process parameters resulted in a highest α-galactosidase activity of 20.4 U/ml. The highest α-galactosidase activity was obtained when the fermentation medium with initial pH 6.0 and containing 1% locust bean gum as growth substrate was inoculated with 10% (v/v) of 72 h grown inoculum and incubated at 30°C. The hydrolysis of flatulence-causing oligosaccharides in soymilk by the enzyme was also investigated. Thin layer chromatographic analysis of enzyme-treated soymilk samples showed the complete hydrolysis of soy oligosaccharides liberating galactose, the final product.     

Ethanol production from corn cob hydrolysates by <Emphasis Type="Italic">Escherichia coli</Emphasis> KO11

Corn cob hydrolysates, with xylose as the dominant sugar, were fermented to ethanol by recombinant Escherichia coli KO11. When inoculum was grown on LB medium containing glucose, fermentation of the hydrolysate was completed in 163 h and ethanol yield was 0.50 g ethanol/g sugar. When inoculum was grown on xylose, ethanol yield dropped, but fermentation was faster (113 h). Hydrolysate containing 72.0 g/l xylose and supplemented with 20.0 g/l rice bran was readily fermented, producing 36.0 g/l ethanol within 70 h. Maximum ethanol concentrations were not higher for fermentations using higher cellular concentration inocula. A simulation of an industrial process integrating pentose fermentation by E. coli and hexose fermentation by yeast was carried out. At the first step, E. coli fermented the hydrolysate containing 85.0 g/l xylose, producing 40.0 g/l ethanol in 94 h. Baker's yeast and sucrose (150.0 g/l) were then added to the spent fermentation broth. After 8 h of yeast fermentation, the ethanol concentration reached 104.0 g/l. This two-stage fermentation can render the bioconversion of lignocellulose to ethanol more attractive due to increased final alcohol concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 124–128 doi:10.1038/sj.jim.7000287 Received 20 February 2002/ Accepted in revised form 04 June 2002     

Semicontinuous lactic fermentation of whey byLactobacillus bulgaricus I. Experimental results

Summary A Monod-like equation correlates the lactic acid productivity and the volume fraction of inoculum in semicontinuous fermentation of whey byLactobacillus bulgaricus. The volume of the inoculum varied from 10% to 80% of the reactor working volume.Nomenclature N number of fermentation cycles after the first fermentation - P lactic acid productivity - S_w average total sugars concentration of the whey as lactose (the standard deviation is indicated) - T average fermentation time (the standard deviation is indicated) - V_a average total consumption of NH₄OH solution (the standard deviation is indicated) - V_i volume of recently fermented medium used as inoculum of the next fermentation cycle - V_w volume of whey added to the reactor at the beginning of each fer mentation cycle - volume fraction of inoculum=V_i/(V_i+V_w)     

Fermentation of paddy malt mash to ethanol by mixed cultures of Saccharomyces cerevisiae and Zymomonas mobilis ZM4 with penicillin G

Traditional fermentation of paddy malt mash (containing 18.1% w/v dextrose equivalent) to paddy arrack using paddy husk as source of inoculum yielded very low level of ethanol (4.25% v/v). Use of yeast isolates obtained from paddy husk as well as a potent ethanol producer like Zymomonas mobilis ZM4 and their combinations in the fermentation revealed that a combination of an yeast isolate PH 03 (Saccharomyces cerevisiae) and Z. mobilis ZM4 produced synergistically and statistically more ethanol (10.1% v/v) than the individual and other combination of cultures. In this process, addition of penicillin G at a concentration of 20 U/ml rather than heat sterilization, helped retention of the limited amylase activity in the mash for simultaneous saccharification and fermentation over 7 d at 30°C. About 98.5% of the carbohydrate was accountable in the fermentation which yielded 86.7% of the theoretical yield of ethanol, apart from biomass and acids.     

Quantitative changes of the alkaloid complex in a submerged culture ofClaviceps paspali

Claviceps paspali FA produced high concentrations of alkaloid under submerged conditions. Their production was found to depend on the developmental stage and treatment of the filamentous culture inoculum. A medium containing Bacto-peptone with a constant composition of amino acids was selected for the preparation of the inoculum. A two-week fermentation in a synthetic medium with mannitol at 24 ± 1 °C resulted in an increased production of total alkaloids from the original value of 100–200 μg/mL to more than 2 000 μg/mL. Addition of tryptophan did not further increase the production of alkaloids but resulted in changes of the spectrum of some metabolites. 2,3-Dihydroxybenzoic acid accompanied the alkaloids in the fermentation medium. α-Hydroxyethyllysergamide was the predominant component of extracellular alkaloids (80 % in the first days of fermentation). During fermentation the level of this alkaloid continuously decreased while the concentration of the accompanying alkaloids,i.e. lysergamide and the corresponding minor isomers, increased. An erratum to this article is available at .     

Levan production by Zymomonas mobilis cells. Attached to plaited spheres

In this work, an immobilization method for polymer-levan production by a non-flocculating Z mobilis culture was developed. The extent of cell attachment to the stainless steel wire surface, culture growth and product synthesis were described. It was established that during short-term passive immobilization of non-flocculation Z mobilis cells on a stainless steel wire surface, sufficient amounts of biomass for proper levan and ethano fermentation could not be obtained. Adherence of cells was improved by pressing the paste-like biomass within stainless steel spheres knitted from wire with subsequent dehydration. Biomass fixed in metal spheres was used for repeated batch fermentation of levan. The activation period of cells within wire spheres (WS) was 48 h in duration. During this time, cell growth stabilized at production levels of ethanol and levan of Q_eth = 1.238 g/l × h and q_eth = 0.47 g/l × h; Q_eth = 0.526 g/l × h and q_eth = 0.20 g/l × h. Five stable fermentation cycles were realized using one wire sphere inoculum, and maintaining a stable ratio of 2.4 of biomass suspended in the medium to biomass fixed in the sphere. Using fixed Z mobilis biomass in the WS, the total amount of inoculum could be reduced for batch fermentation. Large plaited wire spheres with biomass may have potential in fermentation in viscous systems, including levan production.     

A study of the rheological properties of a non-Newtonian fermentation broth

Polysaccharide was synthesized by Aureobasidium pullulans (or Pullularia pullulans) 2552 in a sucrose medium. The field apparent viscosity of the culture medium from shake flask experiments rose to 24,500 cP and then dropped toward its initial value as the fermentation progressed. The magnitude of the maximum apparent viscosity depended on the initial pH of the fermentation broth. The inoculum age influenced the cultivation period before which the maximum viscosity was reached. Rheograms of the fermentation broths showed a change in viscosity behavior from Newtonian to pseudoplastic, and then toward Newtonian characteristics during the fermentation. The calculated non-Newtonian index was found to be a sensitive factor for the indication of the non-Newtonian behavior. Such behavior could not be detected from rheograms. Viscosity profiles of polysaccharide isolated from various stages of the fermentation showed a change from Newtonian to pseudoplastic behavior depending on the concentration (0–2%) of polysaccharide.