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A direct computer control concept for mammalian cell fermentation processes.

In the last 10 years, new assignments and the special demands of mammalian cells to the culture conditions caused the development of complex small scale fermentation setups. The use of continuous fermentation and cell retention devices requires appropriate process control systems. An arrangement for control and data-acquisition of complex laboratory-scale bioreactors is presented. The fundamental idea was the usage of a standard personal computer, which is connected to pumps, valves and sensors via ADA-transformation. The possibility of free programming allowed the development of user-oriented software, especially designed for the far-reaching requirements of a university laboratory in the field of animal cell culture. Control of aeration, pumps, data-acquisition and data-storage are combined within one program, which allows the automation of standard operations like measurement of kLa- or OTR-values. Pump control algorithms for all common fermentation strategies (batch, fed batch, chemostat, perfusion) are included and can be selected any time during cultivation. Oxygen partial pressure and pH are controlled via direct digital control (ddc), providing simple adaption of control parameters and set points to current fermentation conditions.

Animals↗

Yeast communities in a natural tequila fermentation.

Fresh and cooked agave, Drosophila spp., processing equipment, agave molasses, agave extract, and fermenting must at a traditional tequila distillery (Herradura, Amatitan, Jalisco, México) were studied to gain insight on the origin of yeasts involved in a natural tequila fermentations. Five yeast communities were identified. (1) Fresh agave contained a diverse mycobiota dominated by Clavispora lusitaniae and an endemic species, Metschnikowia agaveae. (2) Drosophila spp. from around or inside the distillery yielded typical fruit yeasts, in particular Hanseniaspora spp., Pichia kluyveri, and Candida krusei. (3) Schizosaccharomyces pombe prevailed in molasses. (4) Cooked agave and extract had a considerable diversity of species, but included Saccharomyces cerevisiae. (5) Fermenting juice underwent a gradual reduction in yeast heterogeneity. Torulaspora delbrueckii, Kluyveromyces marxianus, and Hanseniaspora spp. progressively ceded the way to S. cerevisiae, Zygosaccharomyces bailii, Candida milleri, and Brettanomyces spp. With the exception of Pichia membranaefaciens, which was shared by all communities, little overlap existed. That separation was even more manifest when species were divided into distinguishable biotypes based on morphology or physiology. It is concluded that crushing equipment and must holding tanks are the main source of significant inoculum for the fermentation process. Drosophila species appear to serve as internal vectors. Proximity to fruit trees probably contributes to maintaining a substantial Drosophila community, but the yeasts found in the distillery exhibit very little similarity to those found in adjacent vegetation. Interactions involving killer toxins had no apparent direct effects on the yeast community structure.

Alcoholic Beverages↗

Effect of fermentation on the nutritional quality and functional properties of infant food formulations prepared from bambarra-groundnut, fluted-pumpkin and millet seeds.

Weaning foods were formulated by complementation of millet with bambarra groundnut seeds and fluted pumkin seeds. The proximate composition of the individual seeds and the formulations were determined. The composite flour blend containing the highest quantity of fluted pumkin seeds was found to have the highest protein and fat contents (24 and 8% respectively). Functional properties of formulated weaning foods were evaluated. The effect of fermentation on the nutritional quality of the products were determined by animal feeding experiments. The feed intake, weight gain and PER values were evaluated. Fermentation was found to improve the nutritional quality of the formulations. The fermented composite mixture of millet, bambarra groundnut and fluted pumpkin flour (1:1:2) was found to have the highest PER value of 2.1. This suggests that this fermented product will support growth in children better than other formulations.

Animals↗

Nutrient and antinutrient profiles of raw and fermented cocoa beans.

Freshly harvested cocoa bean was subjected to natural fermentation for 3, 6, 9 or 12 days. The proximate, mineral, hydrocyanate (HCN), oxalate and theobromine levels in the products were determined and compared with those of raw (unfermented) cocoa. The latter sample had the following composition: protein (N x 6.25), 17.5; lipid, 62.9; ash, 4.4; fibre, 5.9; and nitrogen-free extract (NFE), 9.3% dry matter. The effect of fermentation was variable, depending on duration and the nutrient under consideration. At day 3, protein content (17.6) was not different, but at day 6 (19.8) was higher (p <0.01), while days 9 and 12 (14.6 and 15.2, respectively) were lower (p <0.01) in comparison with the raw value. Inorganic P steadily decreased from 201.0 (raw) to 102.0 mg/100 g dry matter (day 12) but only the days 9 and 12 values were significantly different (p <O.O5), whereas Ca increased from 29.2 (raw) up to 60.4 mg/100 g dry matter (day 3). The antinutrients, HCN, oxalate, and theobromine decreased with increasing duration of fermentation. The decreases for days 6, 9 and 12 were significant (p <0.05). The results are discussed with regard to optimization of the duration of cocoa fermentation for improved nutrient profile.

Cacao↗

The fermentation of trahanas: a milk-wheat flour combination.

The fermented food, trahanas (a milk-wheat flour combination prepared in Greece), was studied to determine the microbiological and chemical changes that occur during fermentation. It is a lactic acid bacterial fermentation in which Streptococcus lactis, Streptococcus diacetylactis, Leuconostoc cremoris, Lactobacillus lactis, Lactobacillus casei, Lactobacillus bulgaricus and Lactobacillus acidophilus play the major acid- and aroma-producing role. The whole fermentation lasts about 50 hours. The pH of the final dried trahanas was 4.07-4.75, the acidity 0.60-1.00%, the moisture content 8.6-11.5% and the protein content 10.4-13.6%. The product offers possibilities as an increased nutritive value or high-protein food.

Animals↗

Effects of soaking, cooking and fermentation on composition, in-vitro starch digestibility and nutritive value of common beans.

A common bean variety, grown in Burundi, was either fermented, soaked and/or cooked, and then assessed for nutrient composition, in-vitro starch digestibility and protein nutritive value. A decrease in ash, most minerals, vitamins, and some essential amino acids was noted for soaked, cooked and soaked-cooked beans. Compared to untreated beans, soaking decreased soluble sugar (9.8 percent) but increased starch (7.3 percent) and soluble fiber (16.9 percent). In cooked beans, an increase in soluble sugar (1.5 percent), and a decrease in thiamine (81.7 percent), starch (24.6 percent) and soluble fiber (16.6 percent) and nitrogen (2.9 percent) contents were observed. Crude fiber (6.9 percent) and starch (10.0 percent) increased while fat (17.6 percent), fatty acids (linoleic: 10.7 percent; linolenic: 14.3 percent) and soluble sugars (25.4 percent) and nitrogen (14.4 percent) decreased in soaked-cooked beans. Fermentation increased potassium (11.6 percent), soluble fiber (18.9 percent), and some amino acids but decreased fatty acids (linoleic: 13.5 percent; linolenic: 19.9 percent), soluble sugar (75.2 percent) and vitamin (riboflavin: 41.0 percent; niacin: 24.5 percent) contents in common beans. However, the in-vitro starch digestibility was greatly improved (12.3 percent) by cooking while it decreased in soaked beans (29.2 percent). Soaking-cooking and fermentation did not have any significant effect on the digestibility of common bean starch. Finally, among the five treatments applied to common beans, only fermentation showed a significant improvement (8.3 percent) on the protein nutritive value of this legume.

Amino Acids↗

Preparation and fermentation of rice-defatted soy flour blends: effect on protein, fat and ash content.

Rice-defatted soy flour blends prepared in three proportions viz. 40:60, 50:50 and 60:40 were fermented with buttermilk at 25, 30 and 35 degrees C for 12, 18 and 24 h. Fermentation of these blends lowered the pH and raised the titratable acidity, maximum drop in pH and rise in titratable acidity being observed at 35 degrees C for 24 h. The fermentation either decreased or did not change the protein content of cereal-legume blends. Fat as well as ash content remained unaltered irrespective of temperature and time period of fermentation.

Fats↗

Natural fermentation of lentils. Influence of time, concentration and temperature on protein content, trypsin inhibitor activity and phenolic compound content.

Lentil (Lens culinaris var. vulgaris) flour was naturally fermented for 4 days at different temperatures (28 degrees C, 35 degrees C and 42 degrees C) and concentrations (79 milligrams, 150 milligrams and 221 milligrams). Samples were analysed to establish the changes of total protein content and in vitro protein digestibility, trypsin inhibitor activity (TIA) and phenolic compound content during natural fermentation of lentils. The preparation of lentil flour suspensions to be fermented caused a slight increase in total protein and in vitro protein digestibility content, a decrease of TIA and a sharp decrease the tannin/catechin ratio. During the whole fermentation procedure, the minimum initial lentil concentration and temperature used (79 milligrams, 28 degrees C) achieved the maximum protein content and the lowest tannin/catechin ratio. The TIA was more affected by temperature than by concentration, and a 62.5% reduction was observed at 42 degrees C and 79 milligrams.

Catechin↗

Effect of natural fermentation on carbohydrates, riboflavin and trypsin inhibitor activity of lentils.

Lentils were subjected to natural fermentation for 4 days at 30 degrees C. The pH value fell to 3.8 during the process. alpha-Galactosides and sucrose were not detected in fermented lentils but a significant increase in the fructose content was observed. In fermented lentils the neutral detergent fibre, cellulose and hemicellulose contents decreased and the lignin content increased. After fermentation the riboflavin content was higher and the trypsin inhibitor activity decreased.

Carbohydrates↗

Applications of thin layer chromatography, high performance liquid chromatography and mass spectrometry in the fermentation and isolation of the antibiotic nybomycin.

Thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and mass spectrometry (MS) methods have been developed for the analysis of the antibiotic nybomycin, its derivatives deoxynybomycin and nybomycin acetate, during the fermentation and isolation of nybomycin. Using a quantitative HPLC based assay, the time course of nybomycin production (nybomycin titers) in 1000 liter fermentations was determined. Desorption chemical ionization mass spectrometry (DCI/MS) of standard nybomycin samples, fermentation broth samples and purified fractions suggested the co-production of deoxynybomycin which was not reported previously from this organism. TLC and HPLC were used to confirm the presence of deoxynybomycin in the crude extracts of fermentation broths.

Anti-Bacterial Agents↗

Biochemical and physiological characterization of the efrotomycin fermentation.

An efrotomycin fermentation was characterized through physical, chemical and biochemical studies. Growth of the actinomycete, Nocardia lactamdurans occurred during the first 50 h of the fermentation cycle at the expense of glucose, protein, and triglycerides. The initiation of efrotomycin biosynthesis was observed when glucose dropped to a low concentration. Upon glucose depletion, cell growth ceased and a switch in the respiratory quotient occurred. Efrotomycin biosynthesis was supported by the utilization of soybean oil and starch. Analysis of triglyceride metabolism showed that no diglycerides or monoglycerides accumulated during the fermentation. The activity of extracellular enzymes (lipase, protease, and amylase) increased during the cell growth phase and decreased significantly after 150 h. The concentrations of DNA, tetrahydro-vitamin K2 (a membrane component), and free amino acids in the supernatant increased dramatically late in the fermentation cycle (225 h), indicating massive cell lysis. During this same time period, a reduction in cellular respiratory activity and efrotomycin biosynthesis were observed.

Amino Acids↗

The effect of neutral resins on the fermentation production of rubradirin.

Rubradirin is an antibiotic of complex chemical structure which is active vs. methicillin resistant staphylococci. Its development has been limited due to inadequate production yields. The incorporation of neutral resins into fermentations of Streptomyces achromogenes v. rubradiris, UC 8051 resulted in the enhanced production of rubradirin. Resins HP-20, HP-21, XAD-2, XAD-7 and XAD-16 were employed in flask and tank fermentations. The incorporation of these resins promoted 2- to 4-fold enhancements of the rubradirin activity produced in flask fermentations, and the incorporation of XAD-16 and HP-21 into tank fermentations promoted production titer increases greater than 5 fold.

Anti-Bacterial Agents↗

Dose dependency of fermentation and the extent of renal excretion of palatinit (isomalt) in rats with respect to its energy value.

The impact of dose-dependent caloric salvage by microbial fermentation processes in the lower gut and the extent of renal excretion for the overall energetic availability of the alternative bulk sweetener Palatinit were investigated in rats. To evaluate the extent of dose-dependent fermentation a conventional and a germ-free rat model were used and fecal excretions of Palatinit after intragastric application were compared. Because of the lack of bacterial colonization in the gastrointestinal tract in germ-free rat the difference in fecal excretion of Palatinit between germ-free and conventional rat is mainly due to bacterial fermentation. To determine the amount of renal excretion of Palatinit the urine was collected. The experiments were conducted using different amounts of Palatinit (300 and 1,200 mg/kg body weight = mg/kg b.w.). Fecal excretions of Palatinit and its monomers (sorbitol and mannitol) were measured by high-performance liquid chromatography (HPLC) and for the determination of renal excretions a gas chromatography system was used. After the application of 300 mg/kg b.w. Palatinit only the breakdown product sorbitol could be recovered in the feces of germ-free rats (29% of the applied dose). No intact Palatinit could be found. In contrast, neither Palatinit nor the breakdown products sorbitol or mannitol could be detected in the feces of conventional rats after application of the same dose. After the application of the higher dose only small amounts of intact Palatinit were found in the feces of germ-free rats (average 12%). There was no intact measurable Palatinit in the feces of conventional rats. The fecal excretions of sorbitol and mannitol in the feces of the germ-free rats were 55% and 39%; in conventional rats only 21% sorbitol was excreted. Only traces of Palatinit, sorbitol or mannitol were found in the urine of conventional and germ-free rats after application of the low as well as the high dose. In conclusion, this study clearly shows the dose dependency of fermentation and therefore the dose dependency of the energetic (i.e., caloric) availability of this disaccharide sugar alcohol. In the calculation of the energy value of Palatinit the renal excretion of Palatinit and its monomers can be neglected.

Animals↗

Effect of germination and fermentation on the nutritional quality of bambara nut (Voandzeia subterranea L. Thouars) and its product (milk).

Five cultivars of bambara groundnut were selected on which to study the effects of germination and fermentation on their proximate composition. The cultivar that had the highest protein content was chosen to prepare unfermented and fermented milk. Standard assay techniques were adopted to determine the parameters selected for use. Germination caused a decrease in the protein, carbohydrate and starch; it increased sugar content, and had varied effects on the lipids contents of the dry samples. The anti-nutritional factor-tannin concentration was decreased. Germination and fermentation had varied effects on the nutrient compositions of the milk. Compared to the control, germination had the same effect as in the seeds. Fermentation further decreased some of the nitrogenous constituents, sugar and starch content of the milk and much more drastically the tannin content. Hydrolysis and other metabolic changes freed the nutrients from their bound forms while decreasing the quantity, but increasing the quality and availability of the nutrients.

Ammonia↗

Biological utilisation of pearl millet flour fermented with yeasts and lactobacilli.

Mixed culture fermentation of pearl millet flour with Saccharomyces diastaticus, Saccharomyces cerevisiae, Lactobacillus brevis and Lactobacillus fermentum brought about an improvement in its biological utilisation in rats. Protein efficiency ratio, feed efficiency ratio, apparent protein digestibility, true protein digestibility, net protein utilisation, net protein retention, protein retention efficiency and utilisable protein values in case of pure culture fermented pearl millet four were higher than the control. Feeding of the fermented products did not bring about physiological, histopathological and haematological changes in rats. Chapaties and cutlets prepared from the fermented products were organoleptically acceptable.

Animals↗

Sequential fermentation of pearl millet by yeasts and lactobacilli--effect on the antinutrients and in vitro digestibility.

Sequential culture fermentation by yeasts (S. diastaticus or S. cerevisiae) at 30 degrees C for 72 hr and then followed by lactobacilli fermentation (L. brevis or L. fermentum) at 30 degrees C for 72 h more resulted in a significant reduction in phytic acid and polyphenol content of pearl millet flour. Fermentation by S. diastaticus and L. brevis combination almost eliminated phytic acid from pearl millet flour. The combinations of S. diastaticus with both the lactobacilli reduced phytic acid more effectively than those of S. cerevisiae. The products fermented by S. cerevisiae and L. brevis and by S. diastaticus and L. brevis combinations had the highest protein and starch digestibility (in vitro).

Dietary Carbohydrates↗

Production and properties of three pectinolytic activities produced by Aspergillus niger in submerged and solid-state fermentation.

Three extracellular pectinases were produced by Aspergillus niger CH4 by submerged and solid-state fermentation, and their physicochemical and kinetic properties were studied. The highest productivities of endo- and exo-pectinase and pectin lyase were obtained with solid-state fermentation. The kinetic and physicochemical properties of these enzymes were influenced by the type of culture method used. All activities were very different in terms of pH and temperature optima, stability at different pH and temperature values and affinity for the substrate (Km values). In solid-state fermentation, all pectinase activities were more stable at extreme pH and temperature values but the Km values of endo-pectinase and pectin lyase were higher with respect to those activities obtained by the submerged-culture technique. The pectin lyase activity obtained by the submerged-culture technique showed substrate inhibition but the enzyme obtained by solid-state fermentation did not. Electrophoresis, using sodium dodecyl sulphate/polyacrylamide gel with enzymatic extracts obtained for both culture methods, showed the same number of protein bands but some differences were found in their electrophoretic position. The results obtained in this work suggest that the culture method (submerged or solid-state) may be responsible for inducing changes in some of the pectinolytic enzymes produced by A. niger.

Aspergillus niger↗

Studies on the effects of selenium on rumen microbial fermentation in vitro.

The effects of selenium (Se) on ruminant microbial fermentation were investigated in vitro using rumen microflora collected from a rumen-fistulated dairy cow. First, the effects of L-selenomethionine (SeMet; at 0.2 or 2 ppm Se) in the presence or absence of wheat bran (WB, 500 mg per incubation flask) were evaluated. Second, the effects of several forms of Se (elemental Se: 50 ppm Se; sodium selenite: 2 ppm Se; SeMet: 2 ppm Se) were compared. Results showed that the amounts of short-chain fatty acids (SCFAs) tended to be increased by SeMet treatment, whereas SeMet in the presence of WB transiently suppressed fermentation. The addition of SeMet tended to increase the production of acetate while reducing the production of butyrate with the without WB supplementation. Among the different Se compounds tested, the amounts of SCFAs were greater with SeMet treatment, which yielded a higher proportion of acetate compared to other treatments. Selenite did not influence the total SCFAs concentrations; however, it increased the relative proportion of butyrate at the expense of acetate. Elemental Se did not significantly affect fermentation. Higher bacterial Se concentrations were observed for selenite than for SeMet. It was concluded that Se supplementation can influence rumen microbial fermentation and that Se compounds differ in this regard.

Acetic Acid↗