Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

Shifts in rhizosphere microbial communities and enzyme activity of Poa alpina across an alpine chronosequence

This study quantifies the influence of Poa alpina on the soil microbial community in primary succession of alpine ecosystems, and whether these effects are controlled by the successional stage. Four successional sites representative of four stages of grassland development (initial, 4 years (non-vegetated); pioneer, 20 years; transition, 75 years; mature, 9500 years old) on the Rotmoos glacier foreland, Austria, were sampled. The size, composition and activity of the microbial community in the rhizosphere and bulk soil were characterized using the chloroform-fumigation extraction procedure, phospholipid fatty acid (PLFA) analysis and measurements of the enzymes beta-glucosidase, beta-xylosidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, acid phosphatase and sulfatase. The interplay between the host plant and the successional stage was quantified using principal component (PCA) and multidimensional scaling analyses. Correlation analyses were applied to evaluate the relationship between soil factors (C-org, N-t, C/N ratio, pH, ammonium, phosphorus, potassium) and microbial properties in the bulk soil. In the pioneer stage microbial colonization of the rhizosphere of P. alpina was dependent on the reservoir of microbial species in the bulk soil. As a consequence, the rhizosphere and bulk soil were similar in microbial biomass (ninhydrin-reactive nitrogen (NHR-N)), community composition (PLFA), and enzyme activity. In the transition and mature grassland stage, more benign soil conditions stimulated microbial growth (NHR-N, total amount of PLFA, bacterial PLFA, Gram-positive bacteria, Gram-negative bacteria), and microbial diversity (Shannon index H) in the rhizosphere either directly or indirectly through enhanced carbon allocation. In the same period, the rhizosphere microflora shifted from a G(-) to a more G(+), and from a fungal to a more bacteria-dominated community. Rhizosphere beta-xylosidase, N-acetyl-beta-glucosaminidase, and sulfatase activity peaked in the mature grassland soil, whereas rhizosphere leucine aminopeptidase, beta-glucosidase, and phosphatase activity were highest in the transition stage, probably because of enhanced carbon and nutrient allocation into the rhizosphere due to better growth conditions. Soil organic matter appeared to be the most important driver of microbial colonization in the bulk soil. The decrease in soil pH and soil C/N ratio mediated the shifts in the soil microbial community composition (bacPLFA, bacPLFA/fungPLFA, G(-), G(+)/G(-)). The activities of beta-glucosidase, beta-xylosidase and phosphatase were related to soil ammonium and phosphorus, indicating that higher decomposition rates enhanced the nutrient availability in the bulk soil. We conclude that the major determinants of the microllora vary along the successional gradient: in the pioneer stage the rhizosphere microflora was primarily determined by the harsh soil environment; under more favourable environmental conditions, however, the host plant selected for a specific microbial community that was related to the dynamic interplay between soil properties and carbon supply. (C) 2004 Elsevier Ltd. All rights reserved.

In vitro evaluation of fibrolytic enzymes as additives for maize (Zea mays L.) silage - I. Effects of ensiling temperature, enzyme source and addition level

A series of experiments was completed to investigate the impact of addition of enzymes at ensiling on in vitro rumen degradation of maize silage. Two commercial products, Depot 40 (D, Biocatalysts Ltd., Pontypridd, UK) and Liquicell 2500 (L, Specialty Enzymes and Biochemicals, Fresno, CA, USA), were used. In experiment 1, the pH optima over a pH range 4.0-6.8 and the stability of D and L under changing pH (4.0, 5.6, 6.8) and temperature (15 and 39 degreesC) conditions were determined. In experiment 2, D and L were applied at three levels to whole crop maize at ensiling, using triplicate 0.5 kg capacity laboratory minisilos. A completely randomized design with a factorial arrangement of treatments was used. One set of treatments was stored at room temperature, whereas another set was stored at 40 degreesC during the first 3 weeks of fermentation, and then stored at room temperature. Silages were opened after 120 days. Results from experiment I indicated that the xylanase activity of both products showed an optimal pH of about 5.6, but the response differed according to the enzyme, whereas the endoglucanase activity was inversely related to pH. Both products retained at least 70% of their xylanase activity after 48 h incubation at 15 or 39 degreesC. In experiment 2, enzymes reduced (P < 0.05) silage pH, regardless of storage temperature and enzyme level. Depol 40 reduced (P < 0.05) the starch contents of the silages, due to its high alpha-amylase activity. This effect was more noticeable in the silages stored at room temperature. Addition of L reduced (P < 0.05) neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents. In vitro rumen degradation, assessed using the Reading Pressure Technique (RPT), showed that L increased (P < 0.05) the initial 6 h gas production (GP) and organic matter degradability (OMD), but did not affect (P > 0.05) the final extent of OMD, indicating that this preparation acted on the rumen degradable material. In contrast, silages treated with D had reduced (P < 0.05) rates of gas production and OMD. These enzymes, regardless of ensiling temperature, can be effective in improving the nutritive quality of maize silage when applied at ensiling. However, the biochemical properties of enzymes (i.e., enzymic activities, optimum pH) may have a crucial role in dictating the nature of the responses. (C) 2003 Elsevier B.V. All rights reserved.

In vitro evaluation of the fermentation properties of galactooligosaccharides synthesised by alpha-galactosidase from Lactobacillus reuteri

Stirred, pH-controlled anaerobic batch cultures were used to evaluate the in vitro utilisation by canine gut microflora of novel alpha-galactooligosaccharides synthesised with an enzyme extract from a canine Lactobacillus reuteri strain. Fructooligosaccharides (FOS), melibiose and raffinose were used as reference carbohydrates for the prebiotic properties of the synthesised oligosaccharide (galactosyl melibiose mixture-GMM). Addition of Lactobacillus acidophilus was used as control for the evaluation of the synbiotic properties of the oligosaccharide with L. reuteri. Populations of predominant gut bacterial groups were monitored over 48 h of batch culture by fluorescent in situ hybridisation, and short-chain fatty acid (SCFA) production was measured. GMM showed a higher increase in bifidobacteria and lactobacilli population number and size as well as a higher decrease in clostridia population number and size compared to the commercial prebiotics (FOS, melibiose, raffinose). This prebiotic effect was further increased by the addition of L. reuteri followed by a change in the SCFA production pattern compared to GMM alone or GMM with L. acidophilus. The observed change in SCFA production was in accordance with the fermentation properties of L. reuteri, suggesting that the novel synbiotic had a significant effect on the canine gut microflora fermentation.

A novel alpha-galactosidase from I'ifidobacterium bifidum with transgalactosylating properties: gene molecular cloning and heterologous expression

A genomic library of Bifidobacterium bifidum (NCIMB 41171) DNA was constructed in Escherichia coli RA11r (melA(-)B(+)) and one alpha-galactosidase encoding gene was isolated. Conceptual translation combined with insertional mutagenesis analysis indicated an open reading frame (ORF) of 759 amino acid (aa) residues encoding an alpha-galactosidase (named as MelA) of 82.8 kDa. Partial purification and characterisation showed that the enzyme had an apparent native molecular mass of a parts per thousand 243 kDa and a subunit size of a parts per thousand 85 kDa. The enzyme belongs to glycosyl hydrolases 36 family with high aa sequence similarities (a parts per thousand 73%) to other known alpha-galactosidases of bifidobacterial origin. Under optimum pH conditions for activity (pH 6.0) and high melibiose concentration (40% w/v), the enzyme was able to form oligosaccharides with degree of polymerisation (DP) a parts per thousand yen3 at higher concentration than DP = 2, with a total yield of 20.5% (w/w).

Modification of cell wall properties in lettuce improves shelf life

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.

Consumer acceptability and sensory profile of cooked broccoli with mustard seeds added to improve chemoprotective properties

Broccoli, a rich source of glucosinolates, is a commonly consumed vegetable of the Brassica family. Hydrolysis products of glucosinolates, isothiocyanates, have been associated with health benefits and contribute to the flavour of Brassica. However, boiling broccoli causes the myrosinase enzyme needed for hydrolysis to denature. In order to ensure hydrolysis, broccoli must either be mildly cooked or active sources of myrosinase, such as mustard seed powder, can be added post-cooking. In this study, samples of broccoli were prepared in six different ways; standard boiling with and without mustard seeds, sous-vide cooking at low temperature (70 °C) and sous-vide cooking at higher temperature (100 ºC) without mustard and with mustard at two different concentrations. The majority of consumers disliked the mildly cooked broccoli samples (70 ºC, 12 min, sous-vide) which had a hard and stringy texture. The highest mean consumer liking was for standard boiled samples (100 ºC, 7 min). Addition of 1% mustard seed powder developed sensory attributes such as pungency, burning sensation, mustard odour and flavour. One cluster of consumers (32%) found mustard seeds to be a good complement to cooked broccoli, however, the majority disliked the mustard-derived sensory attributes. Where the mustard seeds were partially processed, doubling the addition to 2% led to only the same level of mustard flavour and pungency as 1% unprocessed seeds, and mean consumer liking remained unaltered. This suggests that optimisation of the addition level of partially processed mustard seeds may be a route to enhance bioactivity of cooked broccoli without compromising consumer acceptability.

Enzyme assisted extraction of chitin from shrimp shells (Litopenaeus vannamei)

BACKGROUND: Chemical chitin extraction generates large amounts of wastes and increases partial deacetylation of the product. Therefore, the use of biological methods for chitin extraction is an interesting alternative. The effects of process conditions on enzyme assisted extraction of chitin from the shrimp shells in a systematic way were the focal points of this study. RESULTS: Demineralisation conditions of 25C, 20 min, shells-lactic acid ratio of 1:1.1 w/w; and shells-acetic acid ratio of 1:1.2 w/w, the maximum demineralisation values were 98.64 and 97.57% for lactic and acetic acids, respectively. A total protein removal efficiency of 91.10% by protease from Streptomyces griseus with enzyme-substrate ratio 55 U/g, pH 7.0 and incubation time 3 h is obtained when the particle size range is 50-25 μm, which was identified as the most critical factor. The X-ray diffraction and 13C NMR spectroscopy analysis showed that the lower percent crystallinity and higher degree of acetylation of chitin from enzyme assisted extraction may exhibit better solubility properties and less depolymerisation in comparison with chitin from the chemical extraction. CONCLUSION: The present work investigates the effects of individual factors on process yields, and it has shown that, if the particle size is properly controlled a reaction time of 3 h is more than enough for deproteination by protease. Physicochemical analysis indicated that the enzyme assisted production of chitin seems appropriate to extract chitin, possibly retaining its native structure.