Chemicals for preventing moulding in damp hay

Fewer than one third of more than 100 chemicals tested prevented moulding when 0.5% of active ingredient was added to hay containing 35% water or, alternatively, when 0.02% of active ingredient was added with 0.2% propionic acid or ammonium propionate. Chemicals which prevented moulding in these tests when used alone included aliphatic acids with 2–6 carbon atoms, sorbic acid, short chain aldehydes and phenols, propionic acid, fully or half-neutralized with ammonia, and 8-quinolinol and its derivatives. Formic acid, aliphatic acids with 7–9 carbon atoms, longer chain aldehydes and some fungicides delayed moulding for one to two weeks. Only formaldehyde, 8-quinolinol and its derivatives, and p-nitrophenol increased the activity of 0.2% propionic acid. Although the effective doses of propionic acid, ammonium propionate, and propionic acid, half-neutralised with ammonia, were similar, there was evidence that the presence of free acid in the formulation aided redistribution to overcome undertreatment of pockets in the hay. Further tests of 2-phenyl phenol, phenyl propionate and 8-quinolinol showed that the latter was the best candidate for development. Fungi of the Aspergillus glaucus group were first to colonise most tests that failed, indicating their high degree of tolerance of a wide range of chemicals, and their value as test organisms for prospective preservatives.     

Preservation of baled hay with propionic and formic acids and a proprietary additive

Field-baled hay was incompletely preserved using propionic or formic acids. Results were most satisfactory, although moulding by Aspergillus glaucus group was still frequent, when die hay contained less than 30% water and propionic acid was used. However, much more propionic acid was needed than in laboratory experiments probably because poor distribution of acid in the bale, resulted in under-treated pockets of hay where moulds tolerant to propionic acid could grow and then spread to areas that would otherwise have been adequately treated. In Dewar flasks, moulds, particularly Paecilomyces varioti and the A. glaucus group, could spread from untreated hay into hay containing 2% w/w of propionic acid. Addition of 4 or 10% of propionic acid protected adjacent untreated hay from moulding. The concentration of propionic acid necessary to prevent flasks from heating and moulding increased with thickness of the untreated layers and with water content. Redistribution of acid in the bale after treatment was slow and much variation in acid levels occurred even between adjacent 1 cm segments of grass following storage. Spontaneous heating of moulding hay could cause migration of propionic acid in hay, assisting the spread of fungi. A proprietary additive did not prevent moulding at any hay water content tested.     

Laboratory tests of 8-quinolinol as an additive to volatile fatty acids for preserving hay and other feedstuffs

One part of 8-quinolinol in ten parts of propionic acid, either free or part-neutralised with ammonia, halved the acid required for preserving grass and lucerne hays, beans, rape seed and cereal grains containing sufficient moisture to mould. Propionic acid with 8-quinolinol delays moulding and diminishes the rate of spread of mould growth so that maximum temperatures attained are lowered and growth of thermophilic organisms is prevented. The application of 8-quinolinol and propionic acid to grass on cutting with, or without, use of formic acid as desiccant assisted inhibition of moulding especially in conjunction with ammonium propionate treatment after drying. The additive acts by inhibiting the growth of fungi which tolerate and degrade propionic and related fatty acids. The growth of two such fungi, Paecilomyces varioti and Aspergillus glaucus, is inhibited in culture by much smaller concentrations (less than 200 μM or 30 ppm) of 8-quinolinol than are required on hay, which strongly absorbs the additive and makes it less effective.     

Laboratory application of preservatives to hay and the effects of irregular distribution on mould development

Laboratory methods for the uniform treatment of small amounts of hay were developed and applied to test the effect of the irregular distribution of propionic acid preservatives on the moulding of hay. Fresh, frozen, and re-wetted hay behaved similarly in the tests. A plug of as little as 1 g of untreated hay would initiate moulding in otherwise adequately treated hay (packed in glass tubes). Low acid or high moisture content in treated plugs also reduced the efficiency of preservation, but these parameters had a smaller influence than that of the size of an untreated plug.     

Salmonella typhimurium poultry isolate growth response to propionic acid and sodium propionate under aerobic and anaerobic conditions

Propionic acid and its sodium salt have long been used as additives in poultry feed to reduce microbial populations, including Salmonella spp. Propionic acids in poultry feed may have a potential role in inhibiting growth of Salmonella in the chicken intestine. In this study, we determined growth response of a Salmonella typhimurium poultry isolate to propionic acid and sodium propionate under aerobic and anaerobic conditions. Growth rate consistently decreased with the addition of greater concentrations of either propionic acid or sodium propionate. The extent of growth inhibition was much greater with propionic acid than the sodium form. Media pH decreased only with addition of propionic acid. Growth inhibition was more effective under anaerobic growth conditions with either propionic acid or sodium propionate. When determined at the same pH level, growth rate was significantly lowered by addition of 25 mM of either propionate or sodium propionate alone, and also by the decrease in pH levels (P<0.05). These results showed that growth inhibition of S. typhimurium by propionic acid or sodium propionate is greatly enhanced by pH decrease, and to lesser extent by anaerobiosis. We also found that sodium propionate was more inhibitory for growth of S. typhimurium than propionic acid when compared at the same pH levels.     

Moulding of sugar-cane bagasse and its prevention

When fresh sugar-cane bagasse containing about 50% water and 3% sugar was baled and stacked, it quickly heated to over 50°C and remained hotter than 40°C for 50 days. The residual sucrose was utilized by microbial growth and the content of fungal, bacterial and actinomycete spores increased to more than 10⁸/g dry wt. The spores in heated bagasse were mostly of thermophilic actinomycetes and fungi, and included two actinomycetes implicated in bagassosis. Thermoactinomycetes sacchari occurred in 40% of samples examined, some of which yielded up to 5 × 10⁶ colonies/g, while T. vulgaris occurred in 80% of samples, but these rarely yielded more than 10⁵ colonies/g. Other organisms were cellulolytic and caused fibre deterioration. Heating and moulding could be much decreased either by drying to about 25% water content, which halved the spore content after storage, mostly at the expense of the actinomycetes, or by adding 2% by weight of propionic acid, which decreased the spore content to 4 × 10⁶ spores/g or less even after 18 months' storage. Sometimes adding only 0·6% of propionic acid or 2% of propionic acid applied as a 50% aqueous solution had a similar effect. Treatment with propionic acid thus decreased the deterioration of bagasse, permitted its storage between harvests and prevented the hazard of bagassosis to workers.     

Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor

Fed-batch fermentations of glucose by P. acidipropionici ATCC 4875 in free-cell suspension culture and immobilized in a fibrous-bed bioreactor (FBB) were studied. The latter produced a much higher propionic acid concentration (71.8 +/- 0.8 g/L vs. 52.2 +/- 1.1 g/L), indicating enhanced tolerance to propionic acid inhibition by cells adapted in the FBB. Compared to the free-cell fermentation, the FBB culture produced 20-59% more propionate (0.40-0.65 +/- 0.02 g/g vs. 0.41 +/- 0.02 g/g), 17% less acetate (0.10 +/- 0.01 g/g vs. 0.12 +/- 0.02 g/g), and 50% less succinate (0.09 +/- 0.02 g/g vs. 0.18 +/- 0.03 g/g) from glucose. The higher propionate production in the FBB was attributed to mutations in two key enzymes, oxaloacetate transcarboxylase and propionyl CoA: succinyl CoA transferase, leading to the production of propionic acid from pyruvate. Both showed higher specific activity and lower sensitivity to propionic acid inhibition in the mutant than in the wild type. In contrast, the activity of PEP carboxylase, which converts PEP directly to oxaloacetate and leads to the production of succinate from glucose, was generally lower in the mutant than in the wild type. For phosphotransacetylase and acetate kinase in the acetate formation pathway, however, there was no significant difference between the mutant and the wild type. In addition, the mutant had a striking change in its morphology. With a threefold increase in its length and approximately 24% decrease in its diameter, the mutant cell had an approximately 10% higher specific surface area that should have made the mutant more efficient in transporting substrates and metabolites across the cell membrane. A slightly lower membrane-bound ATPase activity found in the mutant also indicated that the mutant might have a more efficient proton pump to allow it to better tolerate propionic acid. In addition, the mutant had more longer-chain saturated fatty acids (C17:0) and less unsaturated fatty acids (C18:1), both of which could decrease membrane fluidity and might have contributed to the increased propionate tolerance. The enhanced propionic acid production from glucose by P. acidipropionici was thus attributed to both a high viable cell density maintained in the reactor and favorable mutations resulted from adaptation by cell immobilization in the FBB.     

Performance and plasma metabolites of dairy calves fed starter containing sodium butyrate, calcium propionate or sodium monensin

This study was conducted to examine the influence of supplementation of sodium butyrate, sodium monensin or calcium propionate in a starter diet on the performance and selected plasma metabolites (plasma glucose, non-esterified fatty acids and β-hydroxybutyrate) of Holstein calves during pre- and post-weaning periods. Twenty-four newborn Holstein calves were housed in individual hutches until 10 weeks of life, receiving water free choice, and fed four liters of milk daily. Calves were blocked according to weight and date of birth, and allocated to one of the following treatments, according to the additive in the starter: (i) sodium butyrate (150 g/kg); (ii) sodium monensin (30 mg/kg); and (iii) calcium propionate (150 g/kg). During 10 weeks, calves received starter ad libitum, while coast cross hay (Cynodon dactylon (L.) pers.) was offered after weaning, which occurred at the 8th week of age. Weekly, calves were weighted and evaluated for body measurements. Blood samples were taken weekly after the fourth week of age, 2 hours after the morning feeding, for determination of plasma metabolites. No differences were observed among treatments for starter or hay intake, BW and daily gain of the animals. Mean concentrations of selected plasma metabolites were similar in calves fed a starter supplemented with sodium butyrate, sodium monensin and calcium propionate. There was significant reduction in the concentrations of plasma glucose as calves aged. The inclusion of sodium butyrate, calcium propionate or sodium monensin as additives in starter feeds resulted in equal animal performance, before and after weaning, suggesting that sodium monensin may be replaced by organic acid salts.     

Butyric acid stimulates rumen mucosa development in the calf mainly by a reduction of apoptosis

In ruminants the Stimulation of papillar growth by butyric acid is well described but effects on mitosis and apoptosis are not known. To clarify the effect of short chain fatty acids three groups of three calves received a basic ration of 100 g hay per day for 6 weeks and additionally milk replacer. From these, two groups were fed with increasing amounts of the salts of either propionic acid (53 to 390 g) or butyric acid up to (54 to 326 g). The control group instead received an additional isocaloric amount of milk replacer. Mitosis was characterized by Ki67 immunoreactivity, apoptosis by a modified TUNEL assay and by electron microscopy. The feeding regimes led to significant differences of papillar length, increasing from 1.0mm (controls) to 2.2 mm (propionic acid) and 4 mm (butyric acid). This enlargement was partly explained by an increased mitotic rate for the two fatty acid groups. The difference between the fatty acid groups was mainly explained by different apoptotic rates which were only one third for butyric acid compared to propionic acid (P < 0.001). In conclusion, butyric acid is a specific inhibitor of ruminal apoptosis in vivo.     

Metabolic perturbation of acrylate pathway in Lactobacillus plantarum

Engineering microbes with heterologous pathway for production of bio-based products has received considerable attention. Reconstituting such non-native pathway in addition to desired product formation often brings an allosteric modulation in enzymes competing at fragile nodes that result in by-product redistribution, in order to retain energy and redox balance. This work, Lactobacillus plantarum engineered with acrylate pathway for propionate production was studied under similar perspectives. Upon expression, the heterologous pathway did not result in propionic acid production under standard glucose concentration of 20?g/L, but 0.01?mM of propionate was formed when grown under low glucose concentration of 1?g/L. Further analysis of secreted metabolites with increased glucose concentration of 10 and 30?g/L remained futile towards propionate formation but showed reorientation in pyruvate metabolism which was related to the control imposed by the host to regulate the hidden constraints caused by gene perturbation. Further, it was ensured that the limitation of supplements did not play any functional role in inhibiting propionic acid synthesis but still followed similar metabolic pattern which was quite unclear though interpreted to certain extent. Thus, the findings gave insights into physiological and metabolic capabilities of Lactobacillus plantarum that at least in principle can be used to enhance the strain performance for increased propionic acid production.     

Water relations and metabolism of propionate in two yeasts from hay

Many yeasts and bacteria were isolated from moist hays (>30% water content) treated with up to 3% propionic acid-based preservatives. Predominant yeasts were Candida guilliermondii var. guilliermondii and Hyphopichia burtonii. Growth of both species was decreased more than 50% in liquid medium containing 54 mmol/l ammonium propionate but some still occurred in 135 mmol/l propionate. Both metabolized between 80 and 85% of 27 mmol/l ammonium propionate in 1% malt broth within four weeks at 25°C. Growth on solid malt extract agar containing ammonium propionate was decreased by decreasing the water availability (water activity, a_w) in the medium. Growth rates were slightly greater when glycerol rather than NaCl was used to alter a_w in the range 0.995 to 0.93. At both 0.995 and 0.95 a_w optimum growth was at pH 6. The significance of these findings with regard to the preservation of moist hay is discussed.     

Research on the ability of propionic acid and vitamin B12 biosynthesis by <Emphasis Type="Italic">Propionibacterium freudenreichii</Emphasis> strain T82

The purpose of this study was to determine the potential for biosynthesis of propionic acid and vitamin B12 by Propionibacterium freudenreichii T82 in a medium containing various sources of carbon (glucose, fructose, and saccharose). These sugars are present in apple pomaces, which are the waste from the production of apple juice. Using statistical analysis design of experiments (DoE), the results allowed us to determine which sugars (carbon sources) exert the most beneficial influence on the biosynthesis of propionic acid and cobalamin. The highest production of propionic acid by the tested bacterial strain was obtained in a medium in which glucose accounted for at least 50% of the available carbon sources. Depending on the culture medium, the concentration of this metabolite ranged from 23 to 40 g/L. P. freudenreichii T82 produced the smallest amount of acid in medium in which the dominant nutrient source was saccharose. The results obtained indicated an inverse relationship between the amount of acid produced by the bacteria and vitamin B12 biosynthesis. Because of the high efficiency of propionic acid biosynthesis by P. freudenreichii T82, the prospect of using this strain to obtain propionate with the simultaneous disposal of waste materials (such as apple pomaces) which contain glucose and/or fructose is very promising.     

Propionic acid production from glycerol by metabolically engineered Propionibacterium acidipropionici

Large amounts of crude glycerol produced in the biodiesel industry can be used as a low-cost renewable feedstock to produce chemicals and fuels. Compared to sugars (sucrose, glucose, xylose, etc.), glycerol has a lower reducing level, which is of benefit to the production of reduced chemicals. In this work, glycerol as the sole carbon source in propionic acid fermentation by metabolically engineered Propionibacterium acidipropionici (ACK-Tet) was studied. It was found that the adapted ACK-Tet mutant could use glycerol for its growth and produced propionic acid at a high yield of 0.54–0.71 g/g, which was much higher than that from glucose (0.35 g/g). In addition, the production of acetic acid in glycerol fermentation was much less than that from glucose. Thus, glycerol fermentation produced a high purity propionic acid with a high propionic acid to acetic acid ratio of 22.4 (vs. 5 for glucose fermentation), facilitating the recovery and purification of propionic acid from the fermentation broth. The highest propionic acid concentration obtained from glycerol fermentation was 106 g/L, which was 2.5 times of the highest concentration (42 g/L) previously reported in the literature.     

Effect of Solvents and Kinetic Parameters on Synthesis of Ethyl Propionate Catalysed by Poly (AAc-co-HPMA-cl-MBAm)-Matrix-Immobilized Lipase of Pseudomonas aeruginosa BTS-2.

Summary A purified alkaline thermo-tolerant bacterial lipase from Pseudomonas aeruginosa BTS-2 was immobilized on a poly (AAc-co-HPMA-cl-MBAm) hydrogel network. The hydrogel showed approximately 95% binding efficiency for lipase (specific activity 1.96 U mg⁻¹). The immobilized enzyme achieved 65.1% conversion of ethanol and propionic acid (100 mM each) into ethyl propionate in n-nonane at 65 °C in 9 h. When alkane of C-chain length lower than n-nonane was used as the organic solvent, the conversion of ethanol and propionic acid into ethyl propionate decreased with a decrease in the log P value of alkanes. The immobilized lipase retained approximately 30% of its original catalytic activity after five cycles of reuse for esterification of ethanol and propionic acid into ethyl propionate at temperature 65 °C in 3 h. Addition of a molecular sieve (3 ?) to the reaction mixture enhanced the formation of ethyl propionate to 89.3%. Moreover, ethanol and propionic acid when taken a molar ratio of 3:1 further promoted the conversion rate to 94%. However, an increase in the molar ratio of propionic acid with respect to ethanol resulted in a decline of ethyl propionate synthesis.     

Growth yield from electrons available from substrates in aerobic- and photoheterotrophic cultures of Rhodopseudomonas sphaeroides S

A balance of electrons available from acetic acid consumed for growth and oxygen uptake in the aerobic- and photoheterotrophic growth of Rhodopseudomonas sphaeroides S on acetate-minimal medium could be realized the same as in the carbon balance. The unmeasured amounts of yeast extract consumed by the cells grown on propionate–yeast extract media were indirectly estimated from the balance equation of electrons available from carbon substrates. The specific consumption rate of the yeast extract increased with an increase in propionate consumption rate in aerobic and photoheterotrophic cultures. Growth yields from acetic acid and from propionic acid plus yeast extract were calculated on the electron level, i.e., Y_X/ave g cell produced/equivalent electrons available from substrate consumed. Y_X/ave values were 5.0 to 5.8 g cell/ave in photoheterotrophic cultures and 2.7 to 3.6 in aerobic–heterotrophic cultures regardless of different medium compositions.     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

Enhanced beta-galactosidase production by high cell-density culture of recombinant Bacillus subtilis with glucose concentration control

Cell concentration, recombinant protein (beta-galactosidase) level, and the specific enzyme expression level were increased from 19 to 184 g/L, 18.3 to 129 U/mL, and 3.2 to 5.7 U/mg protein, respectively, in fed-batch culture of recombinant Bacillus subtilis when glucose concentration was controlled at 1 g/L as compared with those of conventional fed-batch culture. Glucose concentration of the culture broth was monitored by an automatic on-line glucose analyzer and controlled with a moving identification combined with optimal control (MICOC) strategy. When glucose concentrations were controlled at 10, 1, and 0.2 g/L, accumulated propionic acid concentrations and specific enzyme activities were 18.5, 4.4, and 0.6 g/L and 2.9, 5.7, and 7.1 U/mg protein, respectively. The addition of various concentrations of sodium propionate to the growth medium in batch cultures resulted in a drastic decrease in the growth rate with respect to propionate concentration. The propionic acid was shown to be responsible for cell growth inhibition and enzyme activity reduction in fed-batch culture. (c) 1992 John Wiley & Sons, Inc.     

Methylmalonic and propionic acidemias: lipid profiles of normal and affected human skin fibroblasts incubated with [1-14C]propionate

Normal human skin fibroblasts and those from methylmalonic acidemia and propionic acidemia patients were grown in culture. Following incubation with [1-14C]propionate, the major lipid classes in the cells were separated by thin layer chromatography and isolated fractions analyzed by radio gas chromatography for the presence of odd-numbered long-chain fatty acids; the pattern of even-numbered long-chain fatty acids was obtained also. Normal fibroblasts incorporated a small percentage of propionate into odd-numbered fatty acids which were present in all lipids studied. The abnormal cells incorporated a larger amount while maintaining the characteristic ratios of odd-numbered fatty acids found in the normal line. Most of the radioactivity was associated with phospholipids which are the predominant constituents of cell membranes. A characteristic C15/C17 ratio was found for different phospholipids and the triglyceride fraction; pentadecanoic acid was the principal odd-numbered fatty acid utilized in the assembly of complex lipids. Compared to even-numbered long-chain fatty acids the absolute amount of odd-numbered fatty acids was low (1-2%), even in affected cells. An unusual polar lipid fraction was isolated in the course of the study. In the normal cell it contained several unlabeled eicosanoids which were missing from the same fraction of both affected cell lines.     

The sealed storage of moist flax with and without the use of chemical preservatives

The suitability of ensilage as a means of preserving flax was investigated in a series of experiments in which 400 kg round bales of fresh flax were sealed in polyethylene film or plastic wrapped, with or without the addition of formic acid at 2.5 litre t^-1 or formalin at 5.6 litre t^-1 at the time of baling. Plastic wrapping provided a more effective seal than the bags which were easily punctured by the flax roots resulting in moulding and deterioration of the flax. Where the seal was not broken untreated flax underwent a clostridial fermentation and the pH fell to about 4.8. Cellulolytic activity degraded the flax fibre over a period of 3 to 6 months. The addition of formic acid reduced the cellulolytic activity provided the seal was not broken. In an experiment with 4 kg batches of flax of 65%, 40% or 25% MC sealed in polyethylene film, the addition of formic or propionic acids at 20 g kg^-1 DM did not prevent moulding and deterioration, but both NH₃ and SO₂ at 40 g kg^-1 DM preserved the physical structure of the flax. The NH₃ darkened the flax and made it pliable and unscutchable while the SO₂ bleached it and preserved the fibre without microbiological deterioration. The presence of acids on the moist stored flax appeared to inhibit the progress of normal water retting.     

Effects of DL-Malate on In Vitro Forage Fiber Digestion by Mixed Ruminal Microorganisms

The objective of this study was to evaluate the effects of 0, 4, 8, and 12 mM DL-malate on the in vitro mixed ruminal microorganism fermentation of alfalfa hay and Coastal bermudagrass hay. When alfalfa hay was the substrate, 4 and 8 mM DL-malate numerically increased propionate concentration, and 12 mM DL-malate increased (P < 0.10) propionate. All three concentrations of DL-malate decreased (P < 0.05) the acetate:propionate ratio. In Coastal bermudagrass hay fermentations, all three DL-malate concentrations increased (P < 0.05) propionate and decreased (P < 0.05) the acetate:propionate ratio, while 4 and 12 mM DL-malate numerically increased in vitro dry matter disappearance. When mixed ruminal microorganisms were incubated with 6.25 mM DL-lactic acid and alfalfa hay, 8 and 12 mM DL-malate increased (P < 0.05) final pH, and 12 mM DL-malate increased (P < 0.10) propionate and decreased (P < 0.10) the acetate:propionate ratio. DL-Malate treatment had little effect on in vitro dry matter disappearance. Addition of 8 and 12 mM DL-malate to Coastal bermudagrass hay plus DL-lactic acid fermentations increased (P < 0.05) final pH, and 8 mM DL-malate increased (P < 0.10) in vitro dry matter disappearance. Even though DL-malate treatment consistently increased final pH values in fermentations that included DL-lactic acid, there was not a corresponding increase in in vitro dry matter disappearance of either alfalfa hay or Coastal bermudagrass hay in the 48-h batch culture incubations.