Margarida R.G. Maia

Toxicity of unsaturated fatty acids to the biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens

BackgroundHealth-promoting polyunsaturated fatty acids (PUFA) are abundant in forages grazed by ruminants and in vegetable and fish oils used as dietary supplements, but only a small proportion of PUFA finds its way into meat and milk, because of biohydrogenation in the rumen. Butyrivibrio fibrisolvens plays a major role in this activity. The aim of this study was to investigate the mechanisms by which PUFA affect the growth of B. fibrisolvens, how PUFA are metabolized and the metabolic response to growth in the presence of PUFA.ResultsLinoleic acid (LA; cis-9, cis-12-18:2) and α-linolenic acid (LNA; cis-9, cis-12, cis-15-18:3) increased the lag phase of B. fibrisolvens JW11, LNA having the greater effect. Growth was initiated only when the PUFA had been converted to vaccenic acid (VA; trans- 11-18:1). The major fish oil fatty acids, eicosapentaenoic acid (EPA; 20:5(n- 3)) and docosahexaenoic acid (DHA; 22:6(n- 3)), were not metabolized and prevented growth. Cellular integrity, as determined fluorimetrically by propidium iodide (PI) ingression, was affected as much by 18:1 fatty acids, including VA, as 18:2 fatty acids. The methyl esters of LNA, LA, EPA and DHA had no effect on growth or other measurements. The ATP pool decreased by 2/3 when LA was added to growing bacteria, whereas most acyl CoA pools decreased by >96%.ConclusionsIt was concluded that biohydrogenation occurs to enable B. fibrisolvens to survive the bacteriostatic effects of PUFA, and that the toxicity of PUFA is probably mediated via a metabolic effect rather than disruption of membrane integrity.

Influence of fish oil on ruminal biohydrogenation of C18 unsaturated fatty acids

Dietary cis-9, trans-11-conjugated linoleic acid (CLA) is generally thought to be beneficial for human health. Fish oil added to ruminant diets increases the CLA concentration of milk and meat, an increase thought to arise from alterations in ruminal biohydrogenation of unsaturated fatty acids. To investigate the mechanism for this effect, in vitro incubations were carried out with ruminal digesta and the main biohydrogenating ruminal bacterium, Butyrivibrio fibrisolvens. Linoleic acid (LA) or alpha-linolenic acid (LNA) was incubated (1.67 g/l) with strained ruminal digesta from sheep receiving a 50:50 grass hay-concentrate ration. Adding fish oil (up to 4.17 g/l) tended to decrease the initial rate of LA (P=0.025) and LNA (P=0.137) disappearance, decreased (P<0.05) the transient accumulation of conjugated isomers of both fatty acids, and increased (P<0.05) the accumulation of trans-11-18:1. Concentrations of EPA (20:5n-3) or DHA (22:6n-3), the major fatty acids in fish oil, were low (100 mg/l or less) after incubation of fish oil with ruminal digesta. Addition of EPA or DHA (50 mg/l) to pure cultures inhibited the growth and isomerase activity of B. fibrisolvens, while fish oil had no effect. In contrast, similar concentrations of EPA and DHA had no effect on biohydrogenation of LA by mixed digesta, while the addition of LA prevented metabolism of EPA and DHA. Neither EPA nor DHA was metabolised by B. fibrisolvens in pure culture. Thus, fish oil inhibits ruminal biohydrogenation by a mechanism which can be interpreted partly, but not entirely, in terms of its effects on B. fibrisolvens.

Technical note: Stearidonic acid metabolism by mixed ruminal microorganisms in vitro

Dietary supplementation of stearidonic acid (SDA; 18:4n-3) has been considered a possible strategy to increase n-3 unsaturated fatty acid content in ruminant products; however, little is known about its metabolism in the rumen. In vitro batch incubations were carried out with bovine ruminal digesta to investigate the metabolism of SDA and its biohydrogenation products. Incubation mixtures (4.5 mL) that contained 0 (control), 0.25, 0.50, 0.75, 1.00, 1.25, or 1.50 mg of SDA supplemented to 33 mg (DM basis) of commercial total mixed ration based on corn silage, for dairy cows, were incubated for 72 h at 39°C. The content of most fatty acids in whole freeze-dried cultures was affected by SDA supplementation. Branched-chain fatty acids decreased linearly (P < 0.01), and odd-chain fatty acids decreased quadratically (P < 0.01), particularly from 1.00 mg of SDA and above, whereas most C18 fatty acids increased linearly or quadratically (P ≤ 0.04). Stearidonic acid concentrations at 72 h of incubation were very small (<0.6% of total fatty acids and ≤0.9% of added SDA) in all treatments. The apparent biohydrogenation of SDA was extensive, but it was not affected by SDA concentration (P > 0.05). Biohydrogenation followed a pattern similar to that of other C18 unsaturated fatty acids up to 1.00 mg of SDA. Stearic acid (18:0) and vaccenic acid (18:1 trans-11) were the major fatty acids formed, with the latter increasing 9-fold in the 1.00 mg of SDA treatment. At greater inclusion rates, 18:0 and 18:1 trans isomers decreased (P ≤ 0.03), accompanied by increases in unidentified 18:3 and 18:4 isomers (P = 0.02), suggesting that the biohydrogenation pathway was inhibited. The present results clearly indicate that SDA was metabolized extensively, with numerous 18:4 and 18:3 products formed en route to further conversion to 18:2, 18:1 isomers, and 18:0.

The Potential Role of Seaweeds in the Natural Manipulation of Rumen Fermentation and Methane Production

This study is the first to evaluate the effects of five seaweeds (Ulva sp., Laminaria ochroleuca, Saccharina latissima, Gigartina sp., and Gracilaria vermiculophylla) on gas and methane production and ruminal fermentation parameters when incubated in vitro with two substrates (meadow hay and corn silage) for 24 h. Seaweeds led to lower gas production, with Gigartina sp. presenting the lowest value. When incubated with meadow hay, Ulva sp., Gigartina sp. and G. vermiculophylla decreased methane production, but with corn silage, methane production was only decreased by G. vermiculophylla. With meadow hay, L. ochroleuca and S. latissima promoted similar methane production as the control, but with corn silage, L. ochroleuca increased it. With the exception of S. latissima, all seaweeds promoted similar levels of total volatile fatty acid production. The highest proportion of acetic acid was produced with Ulva sp., G. vermiculophylla, and S. latissima; the highest proportion of butyric acid with the control and L. ochroleuca; and the highest proportion of iso-valeric acid with Gigartina sp. These results reveal the potential of seaweeds to mitigate ruminal methane production and the importance of the basal diet. To efficiently use seaweeds as feed ingredients with nutritional and environmental benefits, more research is required to determine the mechanisms underlying seaweed and substrate interactions.

Colour score as a guide for estimating the protein value of corn gluten feed.

BACKGROUND Dry corn gluten feed (CGF) is a raw material commonly included in compound feeds, particularly for ruminant animals. Its colour can vary from yellow-light brown to dark brown. A general assumption is that darker CGF has a low nutritive value for animals due to heat-damaged protein. This study aims to evaluate the use of colour as a practical guide for estimating CGF protein value. RESULTS Results indicate great variability in the physical and nutritional properties among 28 sources of CGF. Principal component factor analysis was used to reduce a large number of variables into fewer numbers of factors. First factor aggregated 1/Z and acid detergent insoluble N content in opposition to X, Y, e(L) , and L(*2). Second factor aggregated a, a(*), neutral detergent fibre and soluble crude protein (CP) contents in opposition to ash and CP contents, and digestible N and in vitro organic matter digestibility. Multiple regression analysis also showed that physical appearance (colour) was related to nutritional properties, stronger relationships being found between a, a(*) and L(*2) scores and digestible N. CONCLUSION Colour could potentially be used to quickly predict the nutritive value of CGF, particularly digestible N, a key parameter when formulating rations.

Simple and Versatile Turbidimetric Monitoring of Bacterial Growth in Liquid Cultures Using a Customized 3D Printed Culture Tube Holder and a Miniaturized Spectrophotometer: Application to Facultative and Strictly Anaerobic Bacteria.

Here we introduce a novel strategy for turbidimetric monitoring of bacterial growth in liquid culture. The instrumentation comprises a light source, a customized 3D printed culture tube holder and a miniaturized spectrophotometer, connected through optical cables. Due to its small footprint and the possibility to operate with external light, bacterial growth was directly monitored from culture tubes in a simple and versatile fashion. This new portable measurement technique was used to monitor the growth of facultative (Escherichia coli ATCC/25922, and Staphylococcus aureus ATCC/29213) and strictly (Butyrivibrio fibrisolvens JW11, Butyrivibrio proteoclasticus P18, and Propionibacterium acnes DSMZ 1897) anaerobic bacteria. For E. coli and S. aureus, the growth rates calculated from normalized optical density values were compared with those ones obtained using a benchtop spectrophotometer without significant differences (P = 0.256). For the strictly anaerobic species, a high precision (relative standard deviation < 3.5%) was observed between replicates up to 48 h. Regarding its potential for customization, this manifold could accommodate further developments for customized turbidimetric monitoring, such as the use of light-emitting diodes as a light source or flow cells.

The intensification of amyloglucosidase-based saccharification by ultrasound

The present report studied the role of ultrasound (US) energy in the amyloglucosidase-based starch hydrolysis using two complementary approaches: (i) in the activity of six commercially-available amyloglucosidases (using soluble starch as substrate), and (ii) in the hydrolysis of four pure starches from different botanical sources. This corresponds to the first systematic evaluation of the role of US in starch hydrolysis mediated by amyloglucosidase, being a consequence of our previous report that assessed the effect of US in the activity of alpha-amylase (LWT - Food Science and Technology 84 (2017) 674-685). Regarding amlyloglucosidases, three enzymes obtained from Aspergillus niger (AN1-AN3), and Spirizyme Achieve (SPA), Spirizyme Fuel (SPF) and Spirizyme Ultra (SPU) were submitted to a Box-Behnken experimental design in order to establish the optimum conditions for their maximum activity. In the presence of US, we found both inactivation and activation, ranging from -88% (AN3) to 699% (SPA). The US promoted the enzyme activity when combined with lower temperatures (40-60 °C), with a marked effect in Spirizyme enzymes. Based on the optimum conditions established by the experimental design, we also evaluated the role of US in the glucose yield resulting from the hydrolysis of pure starches (corn, rice, potato, wheat). In this case, US led to higher glucose yields in all conditions tested. The enhancement factors observed ranged from 1.2 (AN1, rice starch) to 65 (SPA, potato starch) times. We compared these findings with previous reports, which highlighted the role of US in intensifying amyloglucosidase-based saccharification in mild conditions, by simultaneously influencing both enzyme and substrate. Hence, US power has to be fine-tuned for each particular enzyme in order to maximize process intensification.

Unravelling the phytonutrients and antioxidant properties of European Vicia faba L. seeds

For the first time, seven European varieties of Vicia faba L. seeds were investigated for (1) their phytonutrients profile by HPLC-DAD-MS/MS, (2) total phenolic content (TPC), and (3) antioxidant capacity (DPPH and FRAP assays). A wide range of compounds were identified, namely alkaloids, organic acids, terpenoids, jasmonates, and phenolics; these two latter being the most abundant. TPC ranged between 2.62 and 4.3 mg (gallic acid equivalent) g-1 dry weight, for V. faba major variety Belshi and V. faba minor variety Bauska, respectively. The DPPH radical scavenging capacity showed poor correlation (r = 0.550, P = .041) with TPC, suggesting the presence of other antioxidant sources than phenolics. Still, FRAP was positively correlated with TPC (r = 0.709, P < .01) and DPPH (r = 0.819, P < .01). These results elucidated the phytonutrients and antioxidant properties of V. faba L. seeds as functional food sources.