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Biomedical subjects

R A Prins

Publications and source records attributed to R A Prins.

At least 19 recordsLinked to original sources

Characterization of an anaerobic fungus from llama faeces.

An anaerobic fungus was isolated from llama faeces. Based on its morphological characteristics, polyflagellated zoospores, extensive rhizoid system and the formation of monocentric colonies, the fungus is assigned to the genus Neocallimastix. Neocallimastix sp. L2 is able to grow on several poly-, oligo- and monosaccharides. It differs from other Neocallimastix isolates in its inability to ferment inulin. Neocallimastix sp. L2 requires CO2 for growth. In the presence of 100% CO2 in the gas phase glucose is fermented to H2, CO2, formate, acetate, lactate, succinate and ethanol (33.8, 15.4, 74.3, 69.2, 26.7, 8.2, and 28.7 mmol per 100 mmol glucose, respectively). Reduced sulphur compounds can be used as sulphur source and ammonium or amino acids as nitrogen source. The temperature range for glucose fermentation is from 37 to 42 degrees C with an optimum of around 38 degrees C. The pH range for glucose fermentation is from pH 6 to pH 8 with a broad optimum between pH 6.5 and pH 7.5. The zoospores of Neocallimastix sp. L2 contain ribosomal 'globules' and hydrogenosomes. In the kinetosomes of the zoospores spurs, scoops and skirts are visible. In both the rhizoids and the sporangia 'crystal bodies' and hydrogenosomes are present. Mitochondria were not detected in either of these life stages.

Anaerobiosis

Influence of hydrogen-consuming bacteria on cellulose degradation by anaerobic fungi.

The presence of methanogens Methanobacterium arboriphilus, Methanobacterium bryantii, or Methanobrevibacter smithii increased the level of cellulose fermentation by 5 to 10% in cultures of several genera of anaerobic fungi. When Neocallimastix sp. strain L2 was grown in coculture with methanogens the rate of cellulose fermentation also increased relative to that for pure cultures of the fungus. Methanogens caused a shift in the fermentation products to more acetate and less lactate, succinate, and ethanol. Formate transfer in cocultures of anaerobic fungi and M. smithii did not result in further stimulation of cellulolysis above the level caused by H2 transfer. When Selenomonas ruminatium was used as a H2-consuming organism in coculture with Neocallimastix sp. strain L2, both the rate and level of cellulolysis increased. The observed influence of the presence of methanogens is interpreted to indicate a shift of electrons from the formation of electron sink carbon products to H2 via reduced pyridine nucleotides, favoring the production of additional acetate and probably ATP. It is not known how S. ruminantium exerts its influence. It might result from a lowered production of electron sink products by the fungus, from consumption of electron sink products or H2 by S. ruminantium, or from competition for free sugars which in pure culture could exert an inhibiting effect on cellulolysis.

Anaerobiosis

Reisolation of Ruminobacter parvum.

A cellulolytic gram-negative ovoid motile rod (strain GS III) was isolated from an in vitro incubation of ground barley straw in rumen fluid. The anaerobic, non-sporulating, mesophilic organism strongly answered the original description of Ruminobacter parvum (Kaars Sijpesteijn, 1948). The strain GS III fermented pyruvate, D-arabinose, D-xylose, cellobiose, sucrose, maltose, cellulose, dextrin, xylan and pectin. Products from cellobiose were D-lactate, ethanol, acetate, hydrogen and carbon dioxide. A heat-resistant factor in yeast extract that was not a B-vitamin, a metal or one of the volatile fatty acids was required for growth. The mol % G + C was 51.9. The organism was lost after prolonged subculture.

Animals

'Normalization' of germfree mice with anaerobically cultured caecal flora of 'normal' mice.

Germfree (GF) mice were inoculated with a cultured flora from 10(-1), 10(-3), 10(-5), and 10(-7) dilutions of caecal contents from a 'normal' mouse. GF mice associated with a flora of a 'normal' mouse served as controls. The following intestinal parameters were determined: Colonization resistance (CR), Relative caecal weight (RCW), villus:crypt ratio (jejunum and ileum), IgA-producing cells (jejunum and ileum), beta-aspartyl glycine (faeces), volatile and non-volatile fatty acids (caecum) and bile acids (faeces). Only the 10(-1) culture was able to induce similar changes in the GF mice to a 'normal' flora. The GF + 10(-5) and GF + 10(-7) groups deviated markedly from the controls while the GF + 10(-3) group showed in general intermediate values between GF + SPF and GF + 10(-1) on the one hand and GF + 10(-5) and GF + 10(-7) on the other hand. beta-aspartyl glycine was present only in the GF + 10(-7) group. Scanning electron microscopy (SEM) of ileal contents revealed segmented filamentous organisms in the ileum of controls and the GF + 10(-1) group. The faecal flora consisted mainly of fusiform organisms. In the faeces of the 10(-5) and 10(-7) groups increasing amounts of non-bacterial matter were found, while in the faeces of the other groups virtually only bacteria were seen.

Anaerobiosis

[Degradation of cereal straw with lye].

Straw is a plentiful agricultural waste product. It cannot be used as the sole feed for ruminants and it cannot even serve to satisfy maintenance requirements. The rumen microbes are not capable of breaking down the cell walls in cereal straw rapidly enough or to a sufficient extent. To improve the utilization of straw, efforts were made to treat this material by physical, chemical and biological methods. Some of these procedures will only improve the intake of straw by the animal, whereas the primary object continues to be the improvement of digestibility. Alkali treatment of straw is the most suitable method for this purpose. The mode of action of alkali on the cell walls of straw is described in the present paper.

Animal Feed

Characterization of microbial proteolytic enzymes in the rumen.

Up to 43% of the viable bacteria from the rumen of cows fed grass and concentrates grew on a medium containing casein as the main substrate. Proteolytic counts for a cow fed on straw and concentrates or for a hay-fed cow were lower than counts for cows fed grass and concentrates, both in absolute terms and in relation to the total anaerobic count. In crude enzyme preparations derived from the rumen protozoa, amino acid arylamidase (leucine aminopeptidase)-like activity was the main proteolytic activity observed. In enzyme preparations extracted from the rumen bacteria in the presence of Triton X-100, trypsin-like activity was predominant. Amino acid arylamidase- and metal-chelating proteinase-like activity together with lower activities of carboxypeptidase A and B and a very low chymotrypsin-like activity were found as well. Studies with enzyme inhibitors showed that the bacterial trypsin-like activity was largely of the cysteine-protease type in a hay-fed cow, but in addition comprised serine-protease activity in a cow fed grass and concentrates. Total proteolytic activity of the enzymes in the bacterial fraction and the spectrum of proteolytic enzymes were found to vary with the ration.

Animals

Role of DL-lactic acid as an intermediate in rumen metabolism of dairy cows.

The role of DL-lactic acid as an intermediate in the rumen of a Friesian X Holstein dairy cow adapted to a diet of hay ad libitum plus 12 kg of a concentrate mixture was studied in vitro and in vivo. Concentrations of soluble sugars in the rumen fluid became maximal at 30 min postfeeding, but at 90 min no sugars were detectable. The DL-lactate concentration increased very rapidly to about 30 mm at 30 min after feeding, whereas the maximum total VFA concentration was reached 15 min later. More than 80% of the DL-lactate fermented to VFA was converted by Megasphaera elsdenii. Whereas only 16% of L-lactate was fermented to propionate, 75% of the D-lactate was converted to propionic acid. When all soluble sugars had been fermented, the participation of M. elsdenii to lactate fermentation declined and fermentation patterns for D- and L-lactate became similar yielding mostly acetate. Except for a brief period immediately after feeding, DL-lactate did not appear to be an important precursor of VFA in the rumen of a cow adapted to concentrate feeding. DL-lactate may become a more important intermediate in rumen fermentation temporarily when dairy cows are gradually changed from a hay diet to a diet including concentrates. The first 30 d after parturition, when the changeover takes place, is an unstable period, during which the microbial population is changing to fit the new environment.

Animals

Association of germfree mice with intestinal microfloras obtained from "normal" mice.

A cultured microflora obtained from the caecum of a "normal" mouse was given to 4 groups of germfree mice and was supplied 1x, 2x, 3x and 4x respectively at 5-day intervals. Another group received a 10(-7) dilution of the caecal flora while a group associated with an 'SPF' flora served as control. The difference (measured by 8 parameters) between mice supplied with the cultured flora or with a 10(-7) dilution, both given once only, was small. Supplying the flora 3x resulted in more 'normal' mice compared with mice which received the flora once or twice. The caeca of specified-pathogen-free mice contained more bacteria per gram (microscopic bacterial count), less aerobic and anaerobic bacteria per gram (viable counts), while the yield as percentage of the microscopic bacterial count was lower as compared with the group to which a cultured flora was supplied 4 times.

Animals

[Ruminants as breeding animals (author's transl)].

Ruminants can be considered as up graders of the quality of food when they are kept on feeds low in nitrogen and rich in structural carbohydrates such as cellulose. Many feeds and agricultural wastes of no value or unattractive to monogastric animals can be converted into tractive power, milk, wool, meat and offspring by ruminants. To attain levels of production, concentrates which are readily degraded and high in protein are being increasingly fed, which results in improper use of the ruminant, production losses and metabolic disorders due to unstable rumen fermentation. This is also associated with the fact that our domesticated cattle belong to a group of ruminants particularly equipped to deal with roughage. Most other ruminants in the group of the large roughage feeders are even superior to European cattle breeds in digesting poor roughage. When it would become necessary to increase the use of roughages and agricultural lignocellulose wastes in the near future, a large ruminal volume and a high salivation rate should be adopted as criteria of selection in breeding new cattle breeds.

Animal Feed

Regulation of lactate metabolism in the rumen.

The regulation of lactic acid production, the regulation of lactate fermentation and the role of lactate as intermediate in the rumen metabolism was studied. The pH had a pronounced effect on all three processes and therefore buffer capacity of the rumen contents is also described. Starch gave much less rise to lactic acidosis than soluble sugars, as glucose and fructose. Most bacteria grow faster and therefore produce more lactic acid when amino acids and/or soluble proteins are present in the diet. Activity of LDH (lactate dehydrogenase) of mixed rumen microorganisms is regulated by the NADH/NAD(H) balance and the ATP concentration. About 60% of the LDH in mixed rumen microorganisms is fructose-1, 6-diphosphate independent. Megasphaera elsdenii ferments 60 to 80% of the lactate fermented in the rumen of dairy cattle. Lactate accumulates only when the glycolytic flux (hexose units fermented per unit time per microorganism) is high. During adaptation, the glycolytic flux is increased and lactate may accumulate. After adaptation to a certain diet, the number of microorganisms is changed and the glycolytic flux again is normal and lactate is only a minor intermediate in rumen metabolism.

Animals

Kinetic parameters of lactate dehydrogenases of some rumen bacterial species, the anaerobic ciliate Isotricha prostoma and mixed rumen microorganisms.

A number of kinetic parameters of the lactate dehydrogenases of three rumen bacterial species (Peptostreptococcus productus, Propionibacterium acnes and Actinomyces viscosus), the rumen ciliate Isotricha prostoma and mixed rumen microorganisms (MRM) with respect to NADH, pyruvate, fructose-1,6-diphosphate (FDP) as well as the effects of several nucleotide phosphates were studied. Partially purified LDH of Peptostr. productus had the same kinetic parameters as in crude cell free extracts. Values for Km, determined by Michaelis-Menten kinetics with pyruvate as the substrate, were in the same range for all lactate dehydrogenases. After feeding a cow, changes in the apparent Km and Vmax values for NADH of the total LDH activity in MRM were followed. It is suggested that of the factors studied the ratio NADH/NAD(H) and ATP are the most important regulatory factors for the lactate dehydrogenases of mixed rumen microorganisms.

Actinomyces