Conversion of C14-Labeled Substrates to Volatile Fatty Acids by the Rumen Microbiota

The fermentation of uniformly labeled glucose-C¹⁴, glucose-1-C¹⁴, -2-C¹⁴, and -6-C¹⁴, xylose-1-C¹⁴, cellulose-1-C¹⁴, -2-C¹⁴, and -6-C¹⁴, and lactate-2-C¹⁴ by rumen fluids from cows fed all-hay, hay and concentrate (50:50), and all-concentrate diets was investigated. The results obtained suggested that the Embden-Meyerhof glycolytic pathway is the major pathway of hexose utilization, that the major pathway of xylose fermentation involves hexose synthesis, and that the contributions of the nonrandomizing (acrylate) pathway of propionate formation during glucose, xylose, and cellulose fermentations are 4.5, 8.0, and 10.5%, and 24.6, 25.8, and 17.2%, respectively, by rumen fluids from the cows fed all-hay and all-concentrate rations.     

Effects of Long-Chain Fatty Acids on Growth of Rumen Bacteria

The effects of low concentrations of long-chain fatty acids (palmitic, stearic, oleic, and vaccenic) on the growth of seven species (13 strains) of rumen bacteria were investigated. Except for Bacteroides ruminicola and several strains of Butyrivibrio fibrisolvens, bacterial growth was not greatly affected by either palmitic or stearic acids. In contrast, growth of Selenomonas ruminantium, B. ruminicola, and one strain of B. fibrisolvens was stimulated by oleic acid, whereas the cellulolytic species were markedly inhibited by this acid. Vaccenic acid (trans Δ11 18:1) had far less inhibitory effect on the cellulolytic species than oleic acid (cis Δ9 18:1). Inclusion of powdered cellulose in the medium appeared to reverse both inhibitory and stimulatory effects of added fatty acids. However, there was little carry-over effect observed when cells were transferred from a medium with fatty acids to one without. Considerable variation in response to added fatty acids was noted among five strains of B. fibrisolvens. In general, exogenous long-chain fatty acids appear to have little, if any, energy-sparing effect on the growth of rumen bacteria.     

Rates of Production of Individual Volatile Fatty Acids in the Rumen of Lactating Cows

The rumen fermentation rates in individual lactating cows were measured in four different experiments. The results disclosed that the amounts and proportions of volatile acids formed could vary widely. In one case, a marked difference in the proportions of the acids produced arose within the experiment and correlated with a difference in the proportion of methane formed.

The average rate of production per day was 10.5 moles butyric acid, 12.8 moles propionic acid, and 40 moles acetic acid. Manometric estimations of rate gave lower results than those obtained by the zero-time method, due to delay after sampling and to failure of the acids to liberate stoichiometric quantities of carbon dioxide.

For those experiments in which zero-time rates were estimated, the average specific absorption rates, i.e., the amount absorbed per hour per micromole of acid in the rumen, were 0.37 for butyric acid, 0.38 for propionic acid, and 0.26 for acetic acid.

The carbon dioxide, acids, and microbial cells produced in the rumen fermentation are estimated to account for about 90% of the carbon found in the milk and respiratory CO₂ of the cows. The carbon dioxide from the fermentation was about 27% of the carbon dioxide exhaled.

     

Perturbation of Anion Balance during Inhibition of Growth of Escherichia coli by Weak Acids

During inhibition of cell growth by weak acids, there is substantial accumulation of the weak acid anions in the cytoplasm. This study was undertaken to determine the impact of anion accumulation on cellular pools. At pH 6, growth in the presence of 8 mM acetate led to an internal pool of greater than 240 mM acetate anion and resulted in reduced levels of glutamate in the cell, but there were no significant changes in K⁺ and Na⁺ levels. At low osmolarity, the change in the glutamate pool compensated for only a small fraction of the accumulated acetate anion. However, at high osmolarity, glutamate compensated for over half of the accumulated acetate. Recovery of the normal cytoplasmic pH after the removal of acetate was dependent on the synthesis of glutamate.     

Inhibition of Topoisomerases by Fatty Acids

The inhibitory effects of various fatty acids on topoisomerases were examined, and their structure-activity relationships and mechanism of action were studied. Saturated fatty acids (C_6:0 to C_22:0) did not inhibit topoisomerase I, but cis-unsaturated fatty acids (C_16:1 to C_22:1) with one double bond showed strong inhibition of the enzyme. The inhibitory potency depended on the carbon chain length and the position of the double bond in the fatty acid molecule. The trans-isomer, methyl ester and hydroxyl derivative of oleic acid had no or little inhibitory effect on topoisomerases I and II. Among the compounds studied petroselinic acid and vaccenic acid (C_18:1) with a cis-double bond were the potent inhibitors. Petroselinic acid was a topoisomerase inhibitor of the cleavable complex-nonforming type and acted directly on the enzyme molecule in a noncompetitive manner without DNA intercalation.     

Inhibition of Glycolate Oxidase by Fatty Acids

Gelatin, soy protein, lysozyme, succinyl-casein and succinyl-egg albumin were allowed to react with methyl linoleate (ML) at a relative humidity (RH) of 0% at 50°C for 7 days (protein: ML = 1:1). Gel filtration indicated that only gelatin was extensively fragmented. The gelatin was then incubated with ML under various conditions, and changes in the molecular sizes, the gel forming abilities and the chemical characteristics were investigated. The fragmentation of gelatin was increased by decreasing the RH and with the increase in the ratio of ML to protein. The melting point of gel in heating and cooling gelatin was decreased by increasing the fragmentation. The contents of amide and carbonyl groups increased and that of amino group decreased as the reaction progressed at RH 0%, but no change in C-terminal amino acids was observed. Following the reaction at RH 0%, many kinds of amino acid residues of gelatin were damaged, although in our previous paper [Matoba et al.,Agric. Biol. Chem. , 46, 979 (1982)] such was not detected in casein and egg albumin. From the above results, we conclude that gelatin is susceptible to fragmentation by reaction with oxidizing lipids and one possible mechanism of the degradation may be the –N–C– scission of peptide bonds as proposed by Zirlin and Karel [J. Food Sci., 34, 160 (1969)]. Complex reactions other than this scission may also occur.     

Inhibition of Hyaluronidases and Chondroitinases by Fatty Acids

The inhibitory effects of various fatty acids on three hyaluronidases (h-ST, h-SH and h-SD) and four chondroitinases (c-ABC, c-B, c-ACI and c-ACII) were examined, and their structure-activity relationships and mechanism of action were studied. The fatty acids used in this experiment showed various inhibitory activities against the enzymes. None of the fatty acids did not inhibit h-ST and h-SH. The saturated fatty acids (C 10:0 to C 22:0) showed very weak or no inhibition against h-SD, c-ABC, c-B, c-ACI and c-ACII but the unsaturated fatty acids (C 14:1 to C 24:1) with one double bond strongly inhibited the enzymes, and the inhibitory potency increased with increase in carbon chain length of the fatty acids. In contrast, the increase in number of double bonds caused a decrease in inhibitory potency against the enzymes. The position of the double bond and the stereochemistry of the cis - trans form of oleic acid (C 18:1) did not influence the inhibitory potency against the enzymes. Carboxyl and hydroxyl groups in the fatty acid molecule were concerned in the inhibition of c-ACI. Among the fatty acids, eicosatrienoic acid (C 20:3) generally inhibited h-SD, c-ABC, c-B and c-ACI, and nervonic acid (C 24:1) was a potent inhibitor of c-ACII, and the fatty acids inhibited the enzymes in a noncompetitive manner.     

Inhibition of Microbial Lipases by Fatty Acids

Addition of lard or sodium oleate to the medium used for lipase production by Pseudomonas fragi resulted in a decreased accumulation of lipase in the culture supernatant fluid without affecting cell growth. The production and activity of lipase was inhibited by lard, sodium oleate, and the salts of other unsaturated fatty acids. Some divalent cations, Tweens, lecithin, and bovine serum prevented oleate inhibition, but did not reverse it. Similar inhibitory actions were observed with Geotrichum candidum lipase, but not with a staphylococcal lipase or pancreatic lipase. A protective effect by protein in crude enzyme preparations is indicated. The ability of oleate to lower surface tension does not appear to be related to its ability to inhibit lipase.     

Volatile Fatty Acid Requirements of Cellulolytic Rumen Bacteria

A gas chromatographic method was developed which could separate the isomers isovaleric and 2-methylbutyric acid. Subsequent analyses revealed that most commercially available samples of these acids were cross-contaminated; however, one sample of each acid was found to be pure by this criterion. The growth response of seven strains of cellulolytic rumen bacteria (three strains of Bacteroides succinogenes, three strains of Ruminococcus flavefaciens, and one strain of R. albus) to additions of isobutyric, isovaleric, 2-methylbutyric, valeric, and combinations of valeric and a branched-chain acid was determined. Strains of B. succinogenes required a combination of valeric plus either isobutyric or 2-methylbutyric acid. Isovaleric acid was completely inactive. Either isobutyric or 2-methylbutyric acid was required for the growth of R. albus 7. Strain C-94 of R. flavefaciens grew slowly in the presence of any one of the three branched-chain acids, but a combination of isobutyric and 2-methylbutyric acids appeared to satisfy this organism's growth requirements. None of the individual acids or mixtures of straight- and branched-chain acids allowed growth of R. flavefaciens strain C1a which would approach the response obtained from the total mixture of acids. Further work indicated that all three branched-chain acids were required for optimal growth by this strain, although isovaleric acid only influenced the rate of maximal growth. Either 2-methylbutyric or isovaleric acid allowed growth of nearly the same magnitude as that of the positive control for R. flavefaciens B34b. The presence of acetic acid had little influence on the rate or extent of growth of any of the strains except R. albus 7, for which the extent of growth was markedly increased. Determination of the quantitative fatty acid requirements for the three B. succinogenes strains indicated that 0.1 μmole of valeric per ml and 0.05 μmole of 2-methylbutyric per ml permitted maximal growth. However, with isobutyric acid as the branched-chain component, strains A3c and B21a required 0.1 μmole/ml in contrast to S-85 which exhibited optimal growth at the 0.05 μmole/ml level. By use of mixtures of isobutyric and 2-methylbutyric acids, good growth of C-94 was obtained at concentrations of 0.1 and 0.01 μmole/ml, respectively. About 0.3 μmole/ml of each acid was required for satisfactory growth of C1a.     

Volatile and Cellular Fatty Acids of Haemophilus vaginalis

The major volatile acid produced by Haemophilus vaginalis was acetic acid. The absence of propionate, butyrate, and cellular branched-chain fatty acids indicates that H. vaginalis is not related to Propionibacterium or Butyribacterium.     

Chilling Cells Enhances the Bactericidal Action of Fatty Acids on Escherichia coli

[This corrects the article on p. 153 in vol. 31.].     

Effects of Unsaturated Fatty Acids on the Morphogenesis of an Unsaturated Fatty Acid Auxotroph of Escherichia coli

Supplementation of linoleate or linolenate in a culture medium caused abnormal morphogenesis in an unsaturated fatty acid auxotroph of Escherichia coli K-12.     

Changes in Volatile Fatty Acids and Free Amino Acids during Air-Tobacco Stalks

In our searching program for novel sorbicillin related compounds, three novel compounds, spirosorbicillinols A (1), B (2), and C (3), were isolated from the fermentation broth of the USF-4860 strain isolated from a soil sample. The planar structures of compounds 1–3 were determined from spectroscopic evidence and degradation reaction, and that of 1 was the same as that of 2. The relative stereochemistries of compounds 1–3 were determined by ¹H-¹H coupling constants, the elucidation of HMBC and NOESY spectra in detail. 1 and 2 were stereoisomers at C8 position, each other. We propose that compounds 1 and 2 were formed by exo and endo intermolecular Diels-Alder reaction between sorbicillinol as a diene and scytolide (proposed precursor-1) as a dienophile, respectively. Similarly, we propose that compound 3 was formed by an endo intermolecular Diels-Alder reaction between sorbicillinol and proposed precursor-2.     

Role of Volatile Fatty Acids in Development of the Cecal Microflora in Broiler Chickens during Growth

It is known that volatile fatty acids can inhibit growth of species of the family Enterobacteriaceae in vitro. However, whether these volatile fatty acids affect bacterial populations in the ceca of chickens is unknown. Therefore, a study was conducted to investigate if changes in volatile fatty acids in ceca of broiler chickens during growth affect bacterial populations. Results showed that members of the Enterobacteriaceae and enterococci are present in large numbers in 3-day-old broilers and start to decrease when broilers grow older. Lactobacilli are present in large numbers as well in 3-day-old broilers, but they remain stable during the growth of broilers. Acetate, butyrate, and propionate increase from undetectable levels in 1-day-old broilers to high concentrations in 15-day-old broilers, after which they stabilize. Significant negative correlations could be calculated between numbers of Enterobacteriaceae and concentrations of undissociated acetate, propionate, and butyrate. Furthermore, pure cultures of Enterobacteriaceae isolated from the ceca were grown in the presence of volatile fatty acids. Growth rates and maximal optical density decreased when these strains grew in the presence of increasing volatile fatty acid concentrations. It is concluded that volatile fatty acids are responsible for the reduction in numbers of Enterobacteriaceae in the ceca of broiler chickens during growth.     

Mortality of Pratylenchus penetrans by Volatile Fatty Acids from Liquid Hog Manure

As part of our research program assessing the use of liquid hog manure (LHM) to control root-lesion nematodes, Pratylenchus penetrans, a series of acute toxicity tests was conducted to: (i) examine if non-ionized forms of volatile fatty acids (VFA) are responsible for the mortality of P. penetrans exposed to LHM under acidic conditions, (ii) determine if Caenorhabditis elegans can be a surrogate for P. penetrans in screening tests by comparing their sensitivities to VFA, (iii) characterize the nematicidal effect of individual VFA in LHM to P. penetrans, and (iv) determine whether individual VFA in LHM interact in their toxicity to P. penetrans. LHM was significantly (P ≤ 0.05) more toxic to P. penetrans than a mixture of its main VFA components at concentrations of 5% and 10% (vol. VFA or LHM /vol. in buffer). Pratylenchus penetrans was more sensitive to acetic acid than C. elegans, whereas the sensitivity of both nematode species to n-caproic acid was similar. Individual VFA vary in their lethality to P. penetrans. n-valeric acid was the most toxic (LC₉₅= 6.8 mM), while isobutyric acid was the least toxic (LC₉₅ = 45.7 mM). Individual VFA did not interact in their toxicity to P. penetrans, and their effects were considered additive. VFA account for the majority of the lethal effect of LHM to P. penetrans under acidic conditions. Caenorhabditis elegans cannot be used as a surrogate to P. penetrans in toxicity studies using VFA. The efficacy of LHM to control P. penetrans can be evaluated by assessing its VFA content prior to application, and this evaluation is facilitated by the fact that the interaction of individual VFA appears to be simply additive.     

Inhibition of Soybean Lipoxygenase-1 by Sucrose Esters of Fatty Acids

The effect of sucrose esters of fatty acids with carbon numbers from 8 to 16 on soybean Iipoxygenase-1 (L-1) was investigated. The sucrose esters inhibited L-1 and the inhibitory effect increased with an increase in the carbon number of the fatty acid up to 12, being constant above 12. Kinetic studies showed that two molecules of the sucrose ester bound to. L-1 and inhibited it competitively. Based on the behavior of pK_i dependence on the carbon chain length of the sucrose esters, the dodecyl group appeared to be optimum to bind L-1. The results suggest that sucrose esters bind to the catalytic site of L-1 by hydrophobic interaction of the fatty acid and lead to the loss of activity. Sucrose esters, which are widely used as an emulsifier, can be an effective inhibitor of enzymatic lipid peroxidation in food processing.     

Inhibition of Ethylene Production by Fatty Acids in Fruit and Vegetable Tissues

Fatty acids of chain length from C₄ to C₁₂ inhibited ethyleneproduction in wounded albedo tissue of Hassaku (Citrus hassakuHort. ex Tanaka) fruit. Of the fatty acids tested, caprylicacid (C₈) and capric acid (C₁₀) were the most effective. Lauricacid (C₁₂) was less effective, and caproic acid (C₆) and butyricacid (C₄) were the least effective. Caprylic acid at 5 mM markedlyinhibited ethylene production in not only wounded albedo tissueof citrus fruit but also apple (Malus sylvestris Mill.) cortex,tomato (Lycopersicon esculentum Mill.) pericarp, cucumber (Cucumissativus L.) cortex, banana (Musa AAA group Cavendish subgroup)pulp, broccoli (Brassica oleracea L.) floret, spinach (Spinaciaoleracea L.) leaf, lettuce (Lactuca sativa L.) leaf and mungbean (Vigna radiata [L.] Wilczek) hypocotyl. Caprylic acid inhibitedethylene production at the step of conversion of l-aminocyclopropane-l-carboxylicacid to ethylene. The inhibition could be partially relievedby transferring the tissue to caprylic acid-free medium. (Received June 15, 1982; Accepted August 13, 1982)