Ronan F. Power

Antioxidant and antimicrobial properties of some Chinese plant extracts

Sir, Plant extracts have been known since antiquity to possess notable biological activity, including antioxidant, antibacterial and antifungal properties. There is a growing interest in the use of natural products in the human food and animal feed industries as consumer resistance to synthetic additives increases [1,2]. Examples of synthetic additives include antioxidant chemicals, such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), in addition to chemical antimicrobials such as formic and propionic acid. Concern over the safety of use of these additives in the food chain has led to their restricted use in several countries [2–4]. In addition, antimicrobials of plant origin are of interest to the pharmaceutical industry for the control of microbial pathogens. Although the introduction of antibiotics dramatically improved the treatment of bacterial infections, the emergence of antibiotic resistant strains of bacteria has led to the continuing search for useful natural antimicrobials. Natural antioxidants for the prevention of lipid peroxidative damage, which has been implicated in several disorders such as atherosclerosis, carcinogensis and in the ageing process are also becoming important [5–7]. Chinese herbs and spices have been used as dietary supplements for centuries but their modes of action are not well understood. It is thought that the major mode of action may be through an antioxidant or antimicrobial mechanism. The objective of this study was to evaluate the in vitro antioxidant potential and antimicrobial activity of 12 plant extracts obtained from Shangquin Neoland Biotechnology Ltd., Henan, China, namely: goldthread rhizome (rhizoma Captidix trifolia), skullcap root (radix Scutellaria lateriflora), milkvetch root (radix Astragali ), big head root (Bighead atractylodes rhizome), licorice root (from Ning Xia) (radix Glyerrhizae uralensis), licorice root (from Inner Mongolia) (radix Glyerrhizae uralensis), Chinese white peony root (radix Paeonniae alba), tangerine peel (pericardium Citri reticulatae), pine needle (Pinus tabulaeformis carr), medicated leaven (Massa fermentata medicinalis), hawthorn fruit (wild) (Crataegus cuneata) and hawthorn fruit (cultivated) (Crataegus cuneata) and a commercial combination obtained from David Moore Flavours, Ryhall Road, Stamford, UK. This was formulated to contain equal amounts of the following; milkvetch root (radix Astragali ), licorice root (radix Glyerrhizae uralensis), Chinese white peony root (radix Paeonniae alba), tangerine peel (pericardium Citri reticulatae), medicated leaven (Massa fermentata medicinalis), bamboo shoot (Phyliosach s nigra), trichosanthes fruit (Trichosanthes lirilowii maxim) and radish seed (Raphanus sati us). All samples were ground in a mechanical blender prior to extraction. Each sample was subjected to three extraction procedures; water, ethanol or ethanol:water extraction. In all cases, 1 g samples were used for extraction. For water extraction, each sample (1:8 w/v) was boiled for 10 min, centrifuged at 1000×g and the supernatant stored at 4°C essentially as described by Lin and colleagues [8]. Alcohol extracts were prepared by soaking in absolute ethanol (1:8 w/v) for 30 min at ambient temperature with constant agitation, the sample was then centrifuged and stored as above. Ethanol:water (60:40) extracts were prepared by soaking the samples (1:8 w/v) in this solvent at 70°C for 30 min with agitation as described previously [9]. The sample was centrifuged and stored as described above. For minimum inhibitory concentration (MIC) determinations the ethanol:water extracts of the samples were prepared as above, the supernatant was evaporated to * Corresponding author. E-mail address: cduffy@alltech-bio.com (C. Duffy).

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.

Relationship between pH and Medium Dissolved Solids in Terms of Growth and Metabolism of Lactobacilli and Saccharomyces cerevisiae during Ethanol Production

ABSTRACT The specific growth rates of four species of lactobacilli decreased linearly with increases in the concentration of dissolved solids (sugars) in liquid growth medium. This was most likely due to the osmotic stress exerted by the sugars on the bacteria. The reduction in growth rates corresponded to decreased lactic acid production. Medium pH was another factor studied. As the medium pH decreased from 5.5 to 4.0, there was a reduction in the specific growth rate of lactobacilli and a corresponding decrease in the lactic acid produced. In contrast, medium pH did not have any significant effect on the specific growth rate of yeast at any particular concentration of dissolved solids in the medium. However, medium pH had a significant (P < 0.001) effect on ethanol production. A medium pH of 5.5 resulted in maximal ethanol production in all media with different concentrations of dissolved solids. When the data were analyzed as a 4 (pH levels) by 4 (concentrations of dissolved solids) factorial experiment, there was no synergistic effect (P > 0.2923) observed between pH of the medium and concentration of dissolved solids of the medium in reducing bacterial growth and metabolism. The data suggest that reduction of initial medium pH to 4.0 for the control of lactobacilli during ethanol production is not a good practice as there is a reduction (P < 0.001) in the ethanol produced by the yeast at pH 4.0. Setting the mash (medium) with ≥30% (wt/vol) dissolved solids at a pH of 5.0 to 5.5 will minimize the effects of bacterial contamination and maximize ethanol production by yeast.

Effects of dietary supplementation with Yucca schidigera Roezl ex Ortgies and its saponin and non-saponin fractions on rat metabolism.

Yucca schidigera Roezl ex Ortgies, family Lillaceae, was fractionated with butan-1-ol to yield a butanol extractable fraction (BE; saponin fraction) and a non-butanol fraction (NBE; non-saponin fraction). Four groups of eight male rats were allowed ad libitum access to diets supplemented with water (control) or 200 mg x kg(-1) total Y. schidigera (TOT) or 200 mg x kg(-1) of each of the fractions (NBE or BE). The effects of dietary supplementation with the fractions and their interactions in TOT were analyzed according to the factorial experimental design by two-way analysis of variance. All three supplementation groups displayed significantly reduced serum urea levels (P < 0.05). The TOT and NBE fractions were found to significantly increase serum insulin levels (P < 0.01) in the absence of any fluctuations in serum glucose levels. Urea cycle enzyme activities, namely, arginase (EC 3.5.3.1) and argininosuccinate lyase (EC 4.3.2.1), were significantly decreased (P < 0.05) in vivo, although no effect was observed in vitro. Both fractions displayed effects, indicating that the active constituents are present in both fractions.

The effects of dietary supplementation with Yucca schidigera extract or fractions thereof on nitrogen metabolism and gastrointestinal fermentation processes in the rat

Yucca schidigera was fractionated with butan-1-ol, yielding a butanol-extractable (BE) fraction, containing all the in vitro antimicrobial activity, and the aqueous, non-butanol-extractable (NBE) fraction. Four groups of five female rats (12 weeks old) were allowed ad libitum access to diets supplemented with water (control) or 200 mg kg -1 total Y. schidigera (TOT) or its fraction equivalent of NBE or BE for 64 days. The effects of the fractions and their interactions in the TOT treatment were analysed according to the factorial experimental structure by two-way ANOVA. NBE reduced serum urea (-50%, P = 0.019) and ammonia (-46%, P = 0.037) concentrations, serum/urine concentration quotients of urea (-79%, P = 0.009) and ammonia (-57%, P = 0.002). NBE also reduced hindgut acetate/propionate (-12%, P = 0.007) but increased faecal ammonia concentration (+87%, P = 0.039). BE reduced hindgut indoles (-25%, P = 0.023) and interacted synergystically with NBE in the TOT treatment to further reduce hindgut acetate/propionate by 6% (P = 0.006). NBE increased (+27%, P = 0.002) and BE decreased (-57%, P = 0.005) hindgut urease activity levels, resulting in essentially no change (+4%) in the TOT treatment. The in vitro antimicrobial activity of Y. schidigera is an unlikely explanation for most of its effects in vivo because these are caused by NBE and in vitro antimicrobial activity is exclusive to BE. Sarsasapogenin and smilagenin were also exclusive (>98%) to BE and cannot account for the effects of Y. schidigera on N metabolism.

Assessment of the Effects of Nurmi-Type Cultures and a Defined Probiotic Preparation on a Salmonella Typhimurium 29E Challenge In Vivo

The effects of treatment with an undefined commercial Nurmi-type culture (NTC), cultured cecal contents, and a dual-strain probiotic, containing Enterococcus faecalis and Pediococcus pentosaceus, on Salmonella Typhimurium colonization were evaluated in a specific-pathogen-free bird model. Two sets of trials were performed, and each study was arranged as a randomized complete block design with three treatments. Treatments consisted of (i) control, (ii) commercial NTC, and (iii) cultured cecal contents in the first set of trials and (i) control, (ii) defined probiotic, and (iii) cultured cecal contents in the second set. On day 1, birds were administered 1.2 x 10(7) CFU of the appropriate treatment by oral gavage. On day 3, all birds were challenged with 1 x 10(6) CFU of Salmonella Typhimurium 29E (nalidixic acid resistant). Chicks were asphyxiated with argon gas on day 10, and ceca were aseptically removed. Salmonella Typhimurium counts (CFU per milliliter of cecal contents) were determined on brilliant green agar containing 30 mg of nalidixic acid per liter, and CFU counts were log transformed prior to analysis. Cecal pH and volatile fatty acid concentrations were also determined. Data were analyzed by one-way analysis of variance, and means were compared by Tukeys pairwise analysis. Commercial NTC and cultured cecal contents treatments resulted in a significant decrease (P < or = 0.05) in Salmonella Typhimurium 29E colonization, with the NTC offering a higher level of protection. In the second set of trials, the defined probiotic tended to reduce colonization by Salmonella Typhimurium (P = 0.07), while chicks treated with cultured cecal contents displayed a significant decrease (P = 0.03) when compared to the negative control. No significant change was observed in cecal pH or in acetate and propionate concentrations; however, a significant increase in butyrate concentrations in both the cultured cecal contents and defined probiotic treatment groups was observed when compared to the control birds. These observations suggest that defined cultures are less effective Salmonella control agents than are preparations generated from the complete cecal microflora.

Use of Stringent Selection Parameters for the Identification of Possible Selenium-Responsive Marker Genes in Mouse Liver and Gastrocnemius

Selenium is a trace element that, although toxic in higher concentrations, is essential for human and animal health. In this study, we looked at microarray-based gene expression patterns from liver and gastrocnemius tissues in mice fed either a selenium-deficient diet or diets containing sodium selenite, selenomethionine, or a yeast-derived selenium supplement. A p value cutoff of 0.01 was used to identify a select set of selenium-responsive genes that were consistently differentially expressed across three age groups of mice with both ANOVA and t test analyses. A total of 19 gene transcripts were found to be differentially expressed across the three age groups with at least one selenium-deficient/selenium-supplemented diet comparison. Of those 19 genes, 12 had been previously identified as selenoprotein-encoding genes, and four of the genes, Gpx1, Selh, Sep15, and Sepw1, were differentially expressed in both tissues, all three mouse age groups, and all three diet comparisons. Activities associated with non-selenoproteins encoded by selenium-responsive genes included transport and stress response. The selenophosphate synthetase 2 gene Sephs2 in gastrocnemius tissue and the solute carrier gene Slc48a1 in liver tissue, both up-regulated with selenium-deficient diets compared to all three selenium-supplemented diets, are previously overlooked candidates for dietary selenium marker genes.

Characterization of metal–peptide complexes in feed supplements of essential trace elements

Metal chelates with biomolecules are increasingly used in animal supplementation to increase the bioavailability of essential trace elements. However, the transfer of the chelates is not well understood and speciation studies may bring a comprehensive insight to further investigate the biological uptake mechanism(s) implicated. An analytical method was developed for the characterization of the water-soluble metal complexes in animal feed supplements obtained by reaction of a metal salt with a non-GMO soybean enzymatic digest. The method was based on fractionation of the extract by size-exclusion chromatography followed by the analysis of the metal-containing fraction by reversed-phase nanoHPLC with parallel ICP MS and electrospray MS/MS detection. The metal complexes were identified in the mass spectra owing to the Cu characteristic isotopic pattern; the complexation was corroborated by the presence of a peak corresponding to the non-metallated peptide. The study demonstrated the feasibility of SEC-ICP MS to produce characteristic metal (Cu, Zn, Mn, Fe) distribution patterns, which can be of interest to test batch-to-batch reproducibility and to determine the origin of the supplement. The use of the method could be extended to animal feeds prepared using the metal-chelated complexes. Electrospray MS/MS allowed the identification of a number of Cu complexes with peptides. Four different structure conformations were modeled by means of molecular mechanics investigations to assess the chelation stability.

Insight in the transport behavior of copper glycinate complexes through the porcine gastrointestinal membrane using an Ussing chamber assisted by mass spectrometry analysis.

An Ussing chamber study was conducted in order to investigate the transport behavior of copper glycinate complexes through a porcine gastrointestinal membrane. Organic copper complexes such as copper tri- and tetraglycinates (GGG-Cu(II) and GGGG-Cu(II)) were used as model system. In a novel analytical approach the Ussing chamber was combined with mass spectrometry. Therefore, relevant analytical methods based on MALDI-MS and a coupling of capillary electrophoresis to ICP-MS and ESI-MS were developed for the determination of copper complexes in the mucosal and serosal half-chambers. It was found that 86.1+/-8.5% of copper triglycinate but only 20.8+/-9.9% of copper tetraglycinate penetrated the digestive membrane without modification. Furthermore, inorganic copper species were not detected but a new copper complex (m/z 442) was found to be formed in both compartments of the Ussing chamber.

Expression of Saccharomyces cerevisiae endochitinase in Aspergillus awamori

A gene encoding endochitinase activity was isolated by PCR from Saccharomyces cerevisiae NCYC686 and placed under the control of fungal transcriptional elements regulating glucoamylase expression. Following transformation of Aspergillus awamori with this expression construct, heterologous endochitinase was induced in positive transformants by the addition of starch to the growth medium. A series of optimisation and process development studies were then undertaken, the result of which was the production of heterologous endochitinase activity at 343 U g−1 of total protein. It was found also that the yield of enzyme decreased upon scale-up from shake flask to 3 l pilot scale.