Characterization of hop pectins shows the presence of an arabinogalactan-protein

Hop pectins were extracted from spent hops using acid extraction conditions and were characterized chemically. The acid extraction of spent hops resulted in a yield of 2%, containing 59% of polysaccharides. The hop pectins under investigation had a relatively high molecular weight and an intrinsic viscosity comparable to that of commercially available apple and citrus pectins. The low degree of methyl esterification of these pectins implicates that they are mainly suitable for use in calcium gels. The degree of acetylation and the neutral sugar content were relatively high.

A high molecular weight fraction which contained arabinogalactan-proteins was shown to be present in the hop pectin extract after preparative size-exclusion chromatography. Additionally, a fraction with a lower molecular weight was present containing mainly homogalacturonans. The arabinogalactans in the high molecular weight population consisted of (1→3)- and (1→3,6)-linked galactans highly branched with arabinose and galactose side-chains. The protein part of the arabinogalactan-protein (13%) was found to be rich in cystein, threonin, serinin, alanin, and hydroxyprolin. The molecular weight distribution of the hop pectin after degradation with the enzymes endopolygalacturonase plus pectin methyl esterase suggested that the arabinogalactan-protein present in the hop pectin extract was linked to the pectin and that the arabinogalactan-protein itself had a fairly low molecular weight.     

Eleven new microsatellites for hop (Humulus lupulus L.)

We present a new set of 11 polymorphic microsatellite primer sequences for use with Humulus lupulus. Microsatellite‐enriched libraries for GA_n and GT_n types of repeats were produced. Sequencing of 72 clones revealed 42 unique inserts containing microsatellites, out of which 19 primer pairs were designed and microsatellite amplification was tested on 39 wild hops and cultivars. Eleven primer pairs showed single locus amplification with 2–13 alleles, average 7.2, of which 17 unique alleles were discovered. One primer pair amplified too strong stutter bands, one locus was monomorphic and multilocus amplification was obtained with the remaining six primer pairs.     

Isolation and characterization of polymorphic microsatellites for assessment of genetic variation of hops (Humulus lupulus L.)

Hop (Humulus lupulus L.) cultivars are vegetatively propagated and it is difficult to differentiate them during the process of propagation. Fingerprinting with molecular markers based on DNA could be a useful means of identifying different cultivars. Simple sequence repeats, or microsatellite markers, are the most suitable marker for genetic fingerprinting because they are multi‐allelic and co‐dominant. For this purpose, we have developed primers for 10 new polymorphic microsatellite loci that are suitable for genetic fingerprinting in hop.     

New and resurgent insect pests on low trellis hops

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New polymorphic dinucleotide and trinucleotide microsatellite loci for hop Humulus lupulus L

One hundred and thirty-five microsatellite markers were developed for hop Humulus lupulus L. from di- and trinucleotide-enriched libraries. Seventy-eight primers showed amplification in two tested genotypes. Twenty-four loci were further characterized on a population of 34 hop samples and the number of alleles per locus, observed heterozygosity and expected heterozygosity ranged from two to 20 (9.7 on average), from 0.0294 to 0.9412 (0.6234 on average) and from 0.0294 to 0.9170 (0.6720 on average), respectively. These microsatellite markers will be further used for studying population structures and relationships and for identifying important qualitative and quantitative loci of hop.     

Development of preparative and analytical methods of the hop bitter acid oxide fraction and chemical properties of its components

The bitter acids in hops (Humulus lupulus L.) and beer, such as α-, β-, and iso-α-acids, are known to affect beer quality and display various physiological effects. However, these compounds readily oxidize, and the effect of the oxides on the properties of beer or their potential health benefits are not well understood. In this study, we developed a simple preparative method for the bitter acid oxide fraction derived from hops and designated the constituents as matured hop bitter acids (MHBA). HPLC-PDA-ESI/HRMS and MS² revealed that MHBA are primarily composed of α-acid-derived oxides, which possess a common β-tricarbonyl moiety in their structures similar to α-, β-, and iso-α-acids. We also developed a quantitative analytical method of whole MHBA by HPLC, which showed high precision and reproducibility. Using our newly developed method, the concentration of whole MHBA in several commercial beers was evaluated. Our results will promote the study of bitter acid oxides.     

An overview of fungal community diversity in diseased hop plantations

Samples were taken from several hop fields presenting various symptoms. Fifty-nine pure filamentous fungal strains were isolated and identified through genomic DNA preparations, PCR amplification of the ribosomal DNA internal transcribed spacer region and database interrogations. The most frequent genera were Alternaria (16 isolates) and Epicoccum (14 isolates). The ecosystem was shown to be very diverse, since as many as 27 species belonging to 17 genera were recovered. Furthermore, many of the isolated fungi are known to be involved in phytopathogenesis.     

Anti-proliferative properties of prenylated flavonoids from hops (Humulus lupulus L.) in human prostate cancer cell lines

Chalcones xanthohumol (X) and desmethylxanthohumol (DMX), present in hops (Humulus lupulus L.), and the corresponding flavanones isoxanthohumol (IX, from X), 8-prenylnaringenin (8-PN, from DMX), and 6-prenylnaringenin (6-PN, from DMX), have been examined in vitro for their anti-proliferative activity on human prostate cancer cells PC-3 and DU145. X proved to be the most active compound in inhibiting the growth of the cell lines with IC₅₀ values of 12.3±1.1 μM for DU145 and 13.2±1.1 μM for PC-3. 6-PN was the second most active growth inhibitor, particularly in PC-3 cells (IC₅₀ of 18.4±1.2 μM). 8-PN, a highly potent phytoestrogen, exhibited pronounced anti-proliferative effects on PC-3 and DU145 (IC₅₀ of 33.5±1.0 and 43.1±1.2 μM, respectively), and IX gave comparable activities (IC₅₀ of 45.2±1.1 μM for PC-3 and 47.4±1.1 μM for DU145). DMX was the least active compound. It was evidenced for the first time that this family of prenylated flavonoids from hops effectively inhibits proliferation of prostate cancer cells in vitro.     

Diversity of selected hop cultivars detected by fluorescent AFLPs

 The amplified fragment length polymorphism (AFLP) technique was used to assay eight hop cultivars. The application of fluorescent-labelled primers proved to be a valuable tool and substituted radiolabelling. Digestion with the enzymes EcoRI/ MseI and amplification with primers having three selective bases at the 3’end resulted in distinct banding patterns for imaging with a fluorescent scanner. A total of 523 AFLP fragments derived from eight primer combinations were analysed. On average, 18 polymorphisms per combination were displayed. The Saazer “noble” hop cultivars ‘Saazer’, ‘Tettnanger’ and ‘Spalter’ could not be discriminated. The lowest genetic similarity (GS) between lines was computed for the bitter hops ‘Hallertauer Magnum’ and ‘Wye Target’: GS value of 0.89. The high level of genetic similarity of the analysed hop cultivars is discussed. Received: 11 August 1997 / Accepted: 22 August 1997     

Adventitious shoot regeneration in cultures of Humulus lupulus L. (hop) cvs. Brewers Gold and Nugget

 A very efficient protocol for plant regeneration from two commercial Humulus lupulus L. (hop) cultivars, Brewers Gold and Nugget has been established, and the morphogenetic potential of explants cultured on Adams modified medium supplemented with several concentrations of cytokinins and auxins studied. Zeatin at 4.56 μm produced direct caulogenesis and caulogenic calli in both cultivars. Subculture of these calli on Adams modified medium supplemented with benzylaminopurine (4.4 μm) and indolebutyric acid (0.49 μm) promoted shoot regeneration which gradually increased up to the third subculture. Regeneration rates of 60 and 29% were achieved for Nugget and Brewers Gold, respectively. By selection of callus lines, it has been possible to maintain caulogenic potential for 14 months. Regenerated plants were successfully transferred to field conditions. Received: 10 March 1997 / Revision received: 12 November 1997 / Accepted: 22 November 1998     

Preparation of green colorimetric pH sensor using Humulus lupulus L. (common hop) biomolecular extract for sweat monitoring

Novel smart cotton diagnostic assay was developed toward onsite sensing of sweat pH variations for possible medical applications such as drug test and healthcare purposes. Humulus lupulus L. extract was obtained according to previously reported procedure. As reported by high-performance liquid chromatography (HPLC), the extract demonstrated the presence of hop acids, prenylchalcones, and prenylflavanones, which is responsible for the colorimetric changes. The extract was applied to cellulose fibers employing potassium aluminum sulfate as mordant. This was observed by the formation of mordant/xanthohumol nanoparticles onto cotton surface. The absorption spectra and CIE (Commission Internationale de l'Eclairage) Lab screening of the prepared cotton assay showed colorimetric changes in association with hypsochromic shift from 600 nm to 433 nm upon exposure to sweat simulant fluid (pH < 7). The biochromic activity of the xanthohumol-finished cotton depends mainly on the halochromic performance of the xanthohumol chromophore to show a colorimetric switch from yellow to white owing to intramolecular charge transfer in the xanthohumol molecule. No substantial defects were detected in gas-permeability and stiffness of the treated fabrics. Satisfactory fastness was approved for the xanthohumol-dyed diagnostic cotton assay.     

The distribution of hop latent viroid within plants of Humulus lupulus and attempts to obtain viroid-free plants

The detectability of hop latent viroid (HLVd) was investigated in field-grown hop (Humulus lupulus L.; an herbaceous perennial in which all the aerial parts die at the onset of winter) plants, using dot-blot hybridisation. The viroid was readily detected in all aerial tissues in the second half of the growing season but it could not be detected very early in the season. Between early- and mid-season, HLVd was first detected at the base of the new stems and then apparently spread up them as they grew but only became detectable near the tips of the shoots at mid-season, approximately at the time most elongation growth ended and flowering began. Petioles were the most convenient tissues to test, being easy to collect and, relative to leaf lamina tissue, low in inhibitors. Both dot-blot and in situ hybridisation failed to detect HLVd in shoot tips taken from plants grown at two ‘low’ temperatures (10°C and 15°C). Failure to produce any viroid-free plants by in vitro culture from such tips suggested that they did contain viroid but at levels too low to detect by either method. Lower temperatures and smaller explants are now being investigated as means of producing viroid-free plants.     

Evaluation of a damage threshold for two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae), in hop culture

Damage caused by two‐spotted spider mites (Tetranychus urticae) at harvest to yield, quality (measured in percentage α‐acids content) and cone infestation was assessed on hop cvs Hallertauer Magnum, Hallertauer Tradition and Perle. Acaricide‐untreated hop plants with known levels of T. urticae infestation were compared with neighbouring acaricide‐treated plants. Although in 24 of the 36 experimental harvests the untreated hop plants had spider mite infestations of > 100 mites leaf^?1, yields and α‐acids content from the untreated plants were significantly lower than the treated plants in only four instances. However, although mite infestation of cones from untreated hops were significantly higher than acaricide‐treated plants in 27 of the 36 cases, in only one instance did that cause economic loss. Spider mite infestation levels of c. 90 mites leaf^?1 are tolerable at harvest time with little or no risk of causing economic loss to hop growers.     

Cryopreservation of in vitro-grown shoot-tips of hop (Humulus lupulus L.) using encapsulation/dehydration

 A cryopreservation procedure using encapsulation/dehydration was established for shoot-tips obtained from in vitro-grown shoots of hop. After dissection, shoot-tips were encapsulated in medium with alginate and 0.5 M sucrose. Optimal conditions consisted of preculture for 2 days in solid medium with 0.75 M sucrose, or in increasing sucrose concentrations, desiccation for 4 h with silicagel in a flow cabinet (16% water content) followed by rapid freezing and slow thawing. Shoot recovery after freezing 60 min in liquid nitrogen was around 80%. No phenotypical changes were observed in the recovered plants from cryopreserved shoot-tips growing in the field. Received: 20 April 1997 / Revised: 20 February 1998 / Accepted: 1 Dezember 1998     

Within‐plant distribution of Phorodon humuli (Hemiptera: Aphididae) and natural enemies on hops with implications for sampling and management

A field trial was performed on a hop yard throughout 2006, 2007 and 2008 in order to analyze the within‐plant distribution of Phorodon humuli (Schrank) and its natural enemies. The aphid population was higher on the leaves of the middle section of the main stem. Specifically, the population was significantly higher at 2 and 3.25 m than at 6 m during the three years. Natural enemies consisted of aphidophagous predators of which Coccinella septempunctata was recorded in the highest number. Its number was greater where the aphid population was higher, with a significantly higher number of eggs, larvae and adults at 2 and 3.25 m than at 6 m. Lacewing eggs were also frequently observed, with a significantly higher population where the number of aphids was higher. The number of winged aphids was higher at 3.25 m than at 2 m within a plant. Understanding hop aphid distribution within the hop canopy is important in developing sampling programs, in evaluating injury and helps the development of sustainable management practices.     

Histologial Studies on the Root-Knot Nematodes Nonresistant and Resistant Root Apex of Humulus lupulus L.

The nematodes (Meloidogy incognita Chitwood.) invade near the root apex of the hop (Humulus lupulus L.), and usually lie near the meristematic zone. Fallowing their entrance, there are the formation of the giant cells and the consequent continuous divisions and activities of the flat meristematic cells, which result in formation of the nematode galls. Their sizes are also related to the number of nematodes. String beads nematode galls are often formed at the nematode-non resistant root part of the "Comet". The process of nematodes entering and later changing of the cells and tissue is almost the same as the formation of the nematode gall in other plants. Fewer nematodes can also enter the root tip of the highly resistant "Qingdao Dahua", but the subsequent special differentiation of the cells and tissues in the root have greatly restrained the activities of nematodes and ultimately to their death.     

Modulation of breast cancer cell survival by aromatase inhibiting hop (Humulus lupulus L.) flavonoids

Hop flavonoids are being regarded as attractive molecules to prevent or treat certain forms of cancer. Studies have focused mainly on xanthohumol, the most abundant prenylated chalcone existing in hops extract. However, during the production of beer, or after its ingestion, xanthohumol originates different metabolites, among which isoxanthohumol and 8-prenylnaringenin. The aim of this work was to study the effect of the prenylflavonoids xanthohumol, isoxanthohumol and 8-prenylnaringenin on the breast cancer Sk-Br-3 cell line proliferation, apoptosis and activity of the enzyme aromatase (estrogen synthase). Aromatase activity was determined by a tritiated water assay, cell proliferation was assessed by [³H]thymidine incorporation, sulforhodamine B protein measurement and Ki-67 immunostaining and apoptosis was determined by TUNEL. Our results show that all tested prenylflavonoids were able to inhibit aromatase activity and thus, estrogen formation. Additionally, breast cancer cell line proliferation was decreased and apoptosis induced by all three compounds. The presence of 17β-estradiol in treatment medium was able to revert the effect of the prenylflavonoids on cellular proliferation. These observations strengthen the idea that hop flavonoids may have anti-breast cancer effects and shed new light on a possible mechanism of action by which these effects occur, namely through their ability to decrease estrogen synthesis.     

Some effects of hop latent viroid on two cultivars of hop (Humulus lupulus) in the UK

Some effects of infection by hop latent viroid (HLVd) on two commercial plantings of hop, one each of two high alpha-acid cultivars, are described. The cultivar Omega was severely affected: yield of cones and individual cone weights from infected plants were both lower than from uninfected plants by c. 35% and the alpha-acids content (as assessed by HPLC) of the cones c. 30% lower. Wye Northdown was less severely affected: cone weight was 8% less (though this was not statistically significant) and alpha-acids content lower by c. 15%. In both cultivars beta-acids were higher in infected plants which, together with differences in other chemical components, suggested that cones on infected plants had matured, or were maturing, earlier than those on uninfected plants. These effects, together with the previous finding that HLVd occurs frequently in some cultivars (Barbara, Morton & Adams, 1990), suggest that HLVd is a potentially important constraint on hop production in the UK and should be eliminated if possible.