Comparison of Growth Response by Chemostat Cultured and Batch Cultured Clostridium perfringens Cells in Various Food Substrates

A constant source of exponential phase cells of Clostridium perfringens ATCC 3624 could reduce the time required to carry out the protein quality evaluation test of Solberg et al. (1979) to 4 hr. Clostridium perfringens ATCC 3624 was grown in an anaerobic chemostat using the chemically defined medium, R&S, with glucose as the growth limiting nutrient. The dilution rate was set at 0.06 hr^?1, the pH at 7.2, and the temperature at 43°C. In the transition from a batch culture to a continuous culture an initial oscillatory cell density response was observed. In the steady state, which was continued for as long as 50 days, the cells were typical Gram positive rods, occurring singly or in chains as long as 15 rods, which were occasionally without septa. The fermentative and biochemical responses of the cells did not change. No sporulation occurred when the cells were growing in the chemostat, but spores were observed in the glucose free culture effluent after incubation at 37°C for 24 hr. When cells produced in the chemostat, were cultured in complex media they demonstrated a growth response similar to cells which had been grown in a batch culture. In defined medium the generation time for the chemostat cultured cells was decreased approximately 17%. The chemostat cultured cells can be used as inoculum for the C. perfringens protein quality assay.     

THERMAL STABILIZATION OF FOUR MICROBIAL β-GALACTOSIDASES BY HISTIDINE, CASEIN AMINO ACIDS AND CASEIN

Thermal stability of four microbial β-galactosidases was evaluated in the presence of casein, casein amino acids and histidine. The enzymes from Esch-erichia coli, Kluyveromyces marxianus and Streptococcus thermophilus were stabilized up to 60 fold by these additives, but there was little effect on the enzyme from Aspergillus oryzae. Stabilization by casein amino acids and histidine was largely lactose dependent. Casein amino acids were not as effective as casein for any enzyme. For the K. marxianus enzyme, histidine alone was as effective as casein and the stabilizing effect was proportional to the logarithm of the histidine concentration.     

Factors influencing the synthesis of an extracellular proteinase by Enterococcus faecalis subsp. liquefaciens

Studies were conducted on optimum temperature, pH and the requirement for an energy source, amino acids, casein, Zn²⁺ and Ca²⁺ during the synthesis of an extracellular acid proteinase by Enterococcus faecalis var. liquefaciens. Synthesis was monitored using cells grown to mid-logarithmic phase and resuspended at high density in fresh growth medium. Proteinase production was optimal at 30°C and pH 7.0. Proteinase synthesis, being energy-dependent, occurred only in glycolysing cells. The synthesis was high when lactose but not glucose was utilized as a source of energy, indicating that phospho-β-galactosidase gene might probably be located directly upstream the proteinase gene on a plasmid. Good induction of proteinase synthesis could be achieved by 0.2–0.5 % of either yeast extract or tryptic digested casein, perhaps due to its content of a wide variety of free amino acids. Casein was essential for preventing proteinase autolysis and sustaining the enzyme production. Zn²⁺ and Ca²⁺ were required for the formation of an active extracellular proteinase. The synthesis immediately ceased after addition of chloramphenicol or EDTA. EDTA inactivated the preformed proteinase as well. Sodium chloride at a concentration of 6.5 % inhibited both proteinase synthesis and glycolysis.     

Effects of intake and postruminal casein infusion on performance and concentrations of hormones in plasma of lactating cows

Four Holstein cows were utilized in a Latin square design with a factorial arrangement of treatments to examine the interaction between effects of dry matter intake (107 vs. 78% of energy requirements) and postruminal infusions (water vs. 395 g/d casein) on lactational performance, utilization of nitrogen and energy, and plasma concentrations of hormones. Yields of milk and milk protein were decreased by feed restriction and increased by casein infusion with no treatment interactions. Restricting feed intake decreased total nitrogen intake by 143 g/d and resulted in smaller quantities of fecal, absorbed, urinary, milk, and retained nitrogen compared with cows fed ad libitum. Casein infusion increased total nitrogen intake (55 g/d), absorbed nitrogen (54 g/d), urinary nitrogen excretion (28 g/d), and milk nitrogen (13 g/d). Casein by dry matter intake interactions were not significant for nitrogen utilization. Restricting feed intake increased plasma growth hormone and decreased concentrations of insulin and triiodothyronine. Glucagon, prolactin, and thyroxine were not affected by intake. Casein infusion did not affect growth hormone, insulin, prolactin, triiodothyronine, or thyroxine. Increased milk and milk protein yields obtained with casein infusion were apparently not mediated through changes in circulating concentrations of these hormones; however, plasma glucagon was increased by casein infusions.     

Development of a Chemically Defined Medium for Growth of Bifidobacterium animalis

ABSTRACT The growth of Bifidobacterium animalis was tested, under batch and continuous culture conditions, for dependence upon specific nitrogen compounds. The addition of nitrogen bases (adenine, xantine, and guanine) to the medium containing acid-hydrolyzed casein significantly affected the growth of B. animalis , whereas no significant growth was reported when those bases were added to the medium containing only free amino acids; B. animalis was characterized by a μmax of 0.16/h when growth took place in PROLAB medium containing lactose and nitrogenous bases. Our study is potentially important toward design of improved infant milk formulations for healthy newborns.     

AMINO ACID BALANCE IN YEAST FERMENTATION WITH A SYNTHETIC AMINO ACID MIXTURE AS NITROGEN SOURCE

Two brewery yeasts, one bottom- and one top-fermenting strain, were allowed to ferment an 8% glucose solution containing as nitrogen source an amino acid mixture simulating that obtained when yeast was autolysed. The amounts given were approximately twice as high as the expected requirements. After completion of fermentation the total amounts of each amino acid in the whole system, i. e., in medium and yeast, were determined. The results show that the yeast had not taken up amino acids according to its own composition. The amino acids previously found to be rapidly absorbed from brewery wort were present in the whole system in considerably smaller amounts than in the original medium, indicating that these acids had been utilized as a nitrogen souce or for other purposes. The acids which are taken up slowly from brewery wort were present in larger amounts than in the original medium, indicating that they had been synthesized despite the excess in the medium. The two strains showed relatively similar behaviour.     

Growth-enhancing supplements for various species of the genus Bifidobacterium

Various biological materials were tested for their growth-promoting activity of several bifidobacterial species in a synthetic medium containing ample sources of inorganic salts, vitamins, nitrogen, and carbon. It was found that only Bifidobacterium adolescentis and B. longum (ATCC 15708) grew optimally or near optimally in the synthetic medium. All the other bifidobacteria tested grew optimally only in the synthetic medium supplemented with a growth promoter. The best growth promoters for all bacteria were bovine casein digest and yeast extract rather than human milk whey. Other growth promoters, including human and bovine milk wheys, hog gastric mucin, and bovine serum albumin digest were effective with some bacterial species but not with others. Bifidobacteria also grew well when the bovine casein digest (20 mg/ml) was used as the nitrogen source. Only the yeast extract was able to improve growth under these circumstances. The nature of these growth factors has not yet been determined.     

Factors influencing the synthesis of an extracellular proteinase by Enterococcus faecalis subsp. liquefaciens.

Studies were conducted on optimum temperature, pH and the requirement for an energy source, amino acids, casein, Zn2+ and Ca2+ during the synthesis of an extracellular acid proteinase by Enterococcus faecalis var. liquefaciens. Synthesis was monitored using cells grown to mid-logarithmic phase and resuspended at high density in fresh growth medium. Proteinase production was optimal at 30 degrees C and pH 7.0. Proteinase synthesis, being energy-dependent, occurred only in glycolysing cells. The synthesis was high when lactose but not glucose was utilized as a source of energy, indicating that phospho-beta-galactosidase gene might probably be located directly upstream the proteinase gene on a plasmid. Good induction of proteinase synthesis could be achieved by 0.2-0.5% of either yeast extract or tryptic digested casein, perhaps due to its content of a wide variety of free amino acids. Casein was essential for preventing proteinase autolysis and sustaining the enzyme production. Zn2+ and Ca2+ were required for the formation of an active extracellular proteinase. The synthesis immediately ceased after addition of chloramphenicol or EDTA. EDTA inactivated the preformed proteinase as well. Sodium chloride at a concentration of 6.5% inhibited both proteinase synthesis and glycolysis.     

Effect of Sugar Catabolite Repression in Correlation with the Structural Complexity of the Nitrogen Source on Yeast Growth and Fermentation

Biomass and ethanol production by industrial Saccharomyces cerevisiae strains were strongly affected by the structural complexity of the nitrogen source during fermentation in media containing galactose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low galactose concentrations independent of nitrogen supplementation. At high sugar concentrations altered patterns of galactose utilisation were observed. Biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for baking and brewing ale and lager strains. Baking yeast showed improved galactose fermentation performance in the medium supplemented with casamino acids. High biomass production was observed with peptone and casamino acids for the ale brewing strain, however high ethanol production was observed only in the presence of casamino acids. Conversely, peptone was the nitrogen supplement that induced higher biomass and ethanol production for the lager brewing strain. Ammonium salts always induced poor yeast performance. The results with galactose differed from those obtained with glucose and maltose which indicated that supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, suggesting that sugar catabolite repression has a central role in yeast performance in a medium containing nitrogen sources with differing levels of structural complexity.     

Enterotoxin formation by Clostridium perfringens during sporulation and vegetative growth

Growth, sporulation and enterotoxin production have been followed for three different strains of Clostridium perfringens on a vegetative (FTG) medium and a sporulation (DS) medium. Enterotoxin production was followed by use of enzyme-linked immunosorbent assay (ELISA). All strains produced enterotoxin under both vegetative growth and sporulation in spite of the fact that one strain is known to be enterotoxin negative. The presumed negative strain produced about the same amount of enterotoxin on both media (1 ng/ml). The two other strains produced about 1000–2000 times more enterotoxin during sporulation than during vegetative growth.     

Isolation of Casein by Ultrafiltration and Cryodestabilization

Casein was isolated from skim milk by ultrafiltration, storage of the retentate at -8°C for l-4 wk, and centrifuging of thawed samples at 5,000 ×g for 10 min. The extent of cryodestabilization of casein was dependent on the extent of ultrafiltration and storage time at -8°C. Ultrafiltration to a 4× or 6× volume concentration (VCR) ratio resulted in greater than 95% recovery of casein. Casein thus precipitated could be washed with water at 0°C without any significant loss of casein. Removal of lactose caused accelerated casein cryodestabilization in ultrafiltered samples as compared to control samples. Casein precipitated by this method is dispersible in water, whereas acid precipitated casein is not.     

Isolation of the Pichia pastoris PYC1 gene encoding pyruvate carboxylase and identification of a suppressor of the pyc phenotype

We have cloned and characterized a gene encoding pyruvate carboxylase from the methylotrophic yeast Pichia pastoris. Disruption of this gene produced inability to grow in minimal medium with glucose as carbon source and ammonium as nitrogen source. Growth was possible with aspartate or glutamate as nitrogen source. The gene PpPYC1 expressd from its own promoter was able to rescue the phenotype of Saccharomyces cerevisiae mutants devoid of pyruvate carboxylase. In a P. pastoris strain carrying a disrupted PpPYC1 gene we have isolated spontaneous mutants able to grow in non-permissive conditions. In a mutant strain grown in glucose several enzymes sensitive to catabolite repression were derepressed. The strain also had elevated levels of glutamate dehydrogenase (NAD) both in repressed and derepressed conditions. The sequence of the PpPYC1 gene has been entered in the EMBL nucleotide sequence databank: Accession Number Y11106. © 1998 John Wiley & Sons, Ltd.     

Ussing chamber results for amino acid absorption of protein hydrolysates in porcine jejunum must be corrected for endogenous protein

BACKGROUND: Ussing chambers have been used extensively as an ex vivo model to investigate intestinal nutrient absorption. In this study Ussing chambers were used to investigate the absorption of amino acids and peptides in pig jejunal tissue using a highly hydrolysed casein hydrolysate (PeptoPro®), casein and whey protein isolate after they had been digested with pepsin and pancreatin to simulate digestion products in the jejunum. Jejunal tissue was collected from three pigs and mounted into Ussing chambers, equilibrated, and the luminal chamber loaded with one of three test nitrogen sources. Luminal solution samples were taken every 10 min over a 90 min incubation period and the amino acid concentration determined. To determine the endogenous amino acid contribution of the tissue to the luminal solution, Ussing chambers containing no test N source (blanks) were prepared and treated similarly to the Ussing chambers containing the test N sources. RESULTS: The endogenous amino acid contribution was 0.5 mg in the luminal solution at time 0 and increased to 1 mg after 90 min. The mean amino acid absorption after 10 min incubation for the pre‐digested highly hydrolysed casein hydrolysate (16.6%) was significantly (P < 0.05) higher than for the pre‐digested casein (7.6%) and whey protein isolate (6.7%). CONCLUSION: Endogenous amino acids were a quantitatively significant portion of the luminal solution amino acids present in the Ussing chambers containing the test N sources. Ussing chambers may be a suitable tool for studying amino acid absorption of protein hydrolysates but correction for the endogenous amino acid contribution from the intestinal tissue must be made. Copyright © 2009 Society of Chemical Industry     

FORMATION OF HIGHER ALCOHOLS BY VARIOUS YEASTS

The formation of higher alcohols by various yeasts has been studied as a function of the nitrogen level of the medium. Two types of substrate were used. In one ammonium sulphate was the sole nitrogen source and in the other a complex amino acid mixture simulating that present in brewery wort was used. In the former medium, the patterns exhibited by the yeasts were all very much alike, indicating that the formation of higher alcohols through the biosynthetic activities of yeasts is always basically similar. In the medium containing a complex mixture of amino acids, where the higher alcohols can be formed partly or entirely from the exogenous amino acids through Ehrlich's mechanism, widely different patterns were observed, indicating that the utilization of exogenous amino acids as a source of nitrogen and the extent of regulation of biosynthetic pathways vary with species and strain. All the brewery strains seem to be rather similar and no distinct differences between top and bottom fermentation strains could be observed.     

Extracellular α-Amylase Production by Bacillus brevis MTCC 7521

Alpha amylases have various applications in food processing industries, for example, baking, brewing and distillery industries. Studies of the Ca²⁺ independent α-amylase production were carried out by a strain of Bacillus brevis MTCC 7521 isolated from a brick kiln soil. The optimum temperature, pH and incubation period for amylase production were 50°C, 6.0 and 36 h, respectively. The enzyme secretion was at par in the presence of any of the carbon sources (soluble starch, cassava starch and cassava flour). B. brevis produced more amylase in presence of beef extract as nitrogen source in comparison to other organic nitrogen sources (peptone, yeast extract and casein) and asparagine, potassium nitrate, ammonium sulphate, ammonium nitrate and urea reduced the enzyme activity. The addition of Ca²⁺ (10–40 mM) or surfactants (Tween 20, Tween 40, Tween 60, Tween 80, and sodium lauryl sulphate at 0.02% concentration) in culture medium did not result in further improvement in the enzyme production. The purified enzyme had a molecular mass of 205 kDa in native SDS-PAGE.     

Pyruvate decarboxylase: An indispensable enzyme for growth of Saccharomyces cerevisiae on glucose

In Saccharomyces cerevisiae, the structural genes PDC1, PDC5 and PDC6 each encode an active pyruvate decarboxylase. Replacement mutations in these genes were introduced in a homothallic wild-type strain, using the dominant marker genes APT1 and Tn5ble. A pyruvate-decarboxylase-negative (Pdc⁻) mutant lacking all three PDC genes exhibited a three-fold lower growth rate in complex medium with glucose than the isogenic wild-type strain. Growth in batch cultures on complex and defined media with ethanol was not impaired in Pdc⁻ strains. Furthermore, in ethanol-limited chemostat cultures, the biomass yield of Pdc⁻ and wild-type S. cerevisiae were identical. However, Pdc⁻ S. cerevisiae was unable to grow in batch cultures on a defined mineral medium with glucose as the sole carbon source. When aerobic, ethanol-limited chemostat cultures (D = 0·10 h⁻¹) were switched to a feed containing glucose as the sole carbon source, growth ceased after approximately 4 h and, consequently, the cultures washed out. The mutant was, however, able to grow in chemostat cultures on mixtures of glucose and small amounts of ethanol or acetate (5% on a carbon basis). No growth was observed when such cultures were used to inoculate batch cultures on glucose. Furthermore, when the mixed-substrate cultures were switched to a feed containing glucose as the sole carbon source, wash-out occurred. It is concluded that the mitochondrial pyruvate dehydrogenase complex cannot function as the sole source of acetyl-CoA during growth of S. cerevisiae on glucose, neither in batch cultures nor in glucose-limited chemostat cultures.     

Preparation of casein phosphorylated peptides and casein non-phosphorylated peptides using alcalase

Casein was digested with a cheaper enzyme, alcalase, to produce casein phosphorylated peptides and casein non-phosphorylated peptides concurrently. The casein hydrolyzates were separated to the two kinds of peptides by using combined treatment of CaCl₂ and ethanol. Casein phosphorylated peptides and non-phosphorylated peptides constitute some peptides with molecular weight lower than 2509 Da and 2254 Da respectively as determined using size exclusion HPLC, particularly when a degree of hydrolysis of 20% for the casein hydrolyzates was achieved. At the end, the recovery of casein phosphorylated peptides reached 24%. Phosphorus component of casein phosphorylated peptides was found to be 3.08%. The nitrogen recovery of casein non-phosphorylated peptides was about 76%.     

Effects of amino acids and peptides on rumen microbial growth yields

Experiments were conducted using mixed rumen bacterial cultures to determine which amino acids limited growth. Complete amino acid mixtures stimulated microbial growth alone and when added to casein. Amino acid subgroups did not stimulate growth alone or when added to casein or casein hydrolysates. Results were similar whether growth was limited by periodic addition of low amounts of carbohydrate or when higher amounts were added to batch cultures. Little growth occurred with ammonia as sole N source. Addition of 100 mg/L of amino acids and peptides quadrupled growth, peptides at 10 mg/L resulted in higher growth than the corresponding amount of free amino acids. Apparent saturation of growth occurred when 10 mg/L of a complete amino acid mixture or trypticase was added to cultures. The Michaelis constant values for amino acids and trypticase were determined to be .5 and 1.0 mg/L, respectively. Growth was a linear function of amount of carbohydrate fermented with the coefficient of slope increasing with increasing amino acid concentrations. These experiments demonstrate that growth stimulation from amino acids and proteins is due to the number of amino acids provided in a given mixture rather than specific growth limiting amino acids. Rumen bacterial growth is greatly stimulated by amino acids and peptides, with low affinity constant values, allowing good growth in the concentrations of amino acids and peptides found in vivo     

Casein Hydrolysate Produced Using a Formed-in-Place Membrane Reactor

Casein hydrolysate was continuously produced by hydrolysis of bovine casein with Protease Type XXIII (from Aspergillus oryzue) in a pilotscale formed-in-place membrane reactor. A high percentage (>99%) of TCA soluble nitrogen in the hydrolysate (product) was achieved after 45 min at 37°C and pH 7. The product was completely soluble over pH range 2–9. Water sorption increased 4 to 6.5 times at water activity of 0.35–0.95 as compared to intact casein. The immunologically active casein and immunologically active whey protein in the product were reduced 99% and 97%, respectively. Long term operation showed that the membrane reactor maintained steady production of casein hydrolysate longer than 17 hr.