Whole-genome DNA methylome analysis of different developmental stages of the entomopathogenic fungus Beauveria bassiana NCHU-157 by nanopore sequencing.

The entomopathogenic fungus (EPF), Beauveria bassiana, is an important and commonly used EPF for microbial control. However, the role of DNA methylation has not been thoroughly studied. Therefore, the whole genomic DNA methylome of one promising EPF isolate, B. bassiana NCHU-157 (Bb-NCHU-157), was investigated by Oxford Nanopore Technologies (ONT). First, the whole genome of Bb-NCHU-157 was sequenced by next-generation sequencing (NGS) and ONT. The genome of Bb-NCHU-157 contains 16 contigs with 34.19 Mb and 50% GC content, which are composed of 10,848 putative protein-coding genes. Two putative DNA methyltransferases (DNMTs) were found, including Dim-2 and C-5 cytosine-specific DNA methylases. Both DNMTs showed higher expression levels in the mycelium stage than in the conidia stage, indicating that development of DNA methylation in Bb-NCHU-157 might occur in the mycelium stage. The global methylation level of the mycelium stage (5 mC = 4.56%, CG = 3.33%, CHG = 0.74%, CHH = 0.49%) was higher than that of the conidial stage (5 mC = 2.99%, CG = 1.99%, CHG = 0.63%, CHH = 0.37%) in both the gene and transposable element (TE) regions. Furthermore, the TE regions showed higher methylation frequencies than the gene regions, especially for CHH site methylation, suggesting regulation of genomic stabilization during mycelium development. In the gene regions, high methylation frequencies were found around the transcription start site (TSS) and transcription end site (TES). Moreover, CG and CHG methylation mainly occur in the promoter and intergenic regions, while CHH methylation occurs in the TE region. Among the methylated regions, 371, 661, and 756 differentially DNA methylated regions (DMRs) were hypermethylated in the mycelium in CG, CHG, and CHH, while only 13 and 7 DMRs were hypomethylated in the mycelium in CHG, and CHH, respectively. Genes located in the DMR shared the GO terms, DNA binding (GO: 0003677), and sequence-specific DNA binding (GO: 0043565) for hypermethylation in the mycelium, suggesting that methylation might regulate gene expression from the initial process. Evaluation of the DNA methylome in Bb-NCHU-157 by ONT provided new insight into this field. These data will be further validated, and epigenetic regulation during the development of B. bassiana will be explored.

Allergenicity reduction of the bio-elicited peanut sprout powder (BPSP) and toxicological acceptance of BPSP-supplemented diets assessed with ICR mice.

The allergenic and toxicological acceptances of the bio-elicited peanut sprout powder (BPSP) have not been assessed. BPSP was generated from peanut kernels germinated at 26-28 °C for 72 h (designated as 72 h-NGS). The 72 h-NGS were subsequently sliced, incubated, dried, defatted and pulverized to generate bio-elicited peanut sprout powder (BPSP). Protein solubility of BPSP increased 2.6-fold compared to 72 h-NGS. SDS-PAGE analysis revealed BPSP production triggered extensive degradation of the high-molecular weight peanut allergic proteins, mainly Ara h 1 and Ara h 3. Western blotting detected with peanut allergic patients' IgE indicated decreased in vitro reactivity. Food safety assessment of BPSP was performed with ICR mice fed with basal (control) and three doses of formulated BPSP-supplemented diets containing 0.11 g (normal), 2.5 g (high) and 25 g (super high) BPSP /kg BW. Animals appeared healthy with steady body weight gain in all groups during the entire 35-day dietary intervention. Hematological and serum biochemical analyses revealed no significant difference among groups. Histopathological examination on the tissue sections of primary organs further supported safety with no pathologies. The in vitro allergic reduction and toxicological safety in the BPSP-supplemented dietary intervention in the ICR mice study, support moving forward with BPSP-involved product development. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-022-05537-7.

Transcriptome of Nosema ceranae and Upregulated Microsporidia Genes during Its Infection of Western Honey Bee (Apis mellifera).

Nosema ceranae is one of the fungal parasites of Apis mellifera. It causes physical and behavioral effects in honey bees. However, only a few studies have reported on gene expression profiling during A. mellifera infection. In this study, the transcriptome profile of mature spores at each time point of infection (5, 10, and 20 days post-infection, d.p.i.) were investigated. Based on the transcriptome and expression profile analysis, a total of 878, 952, and 981 differentially expressed genes (DEGs) (fold change ≥ 2 or ≤ -2) were identified in N. ceranae spores (NcSp) at 5 d.p.i., 10 d.p.i., and 20 d.p.i., respectively. Moreover, 70 upregulated genes and 340 downregulated genes among common DEGs (so-called common DEGs) and 166 stage-specific genes at each stage of infection were identified. The Gene Ontology (GO) analysis indicated that the DEGs and corresponding common DEGs are involved in the functions of cytosol (GO:0005829), cytoplasm (GO:0005737), and ATP binding (GO:0005524). Furthermore, the pathway analysis found that the DEGs and common DEGs are involved in metabolism, environmental information processing, and organismal systems. Four upregulated common DEGs with higher fold-change values, highly associated with spore proteins and transcription factors, were selected for validation. In addition, the stage-specific genes are highly involved in the mechanism of pre-mRNA splicing according to GO enrichment analysis; thus, three of them showed high expression at each d.p.i. and were also subjected to validation. The relative gene expression levels showed a similar tendency as the transcriptome predictions at different d.p.i., revealing that the gene expression of N. ceranae during infection may be related to the mechanism of gene transcription, protein synthesis, and structural proteins. Our data suggest that the gene expression profiling of N. ceranae at the transcriptomic level could be a reference for the monitoring of nosemosis at the genetic level.

Sacbrood viruses cross-infection between Apis cerana and Apis mellifera: Rapid detection, viral dynamics, evolution and spillover risk assessment.

Recent outbreaks of sacbrood virus (SBV) have caused serious epizootic disease in Apis cerana populations across Asia including Taiwan. Earlier phylogenetic analyses showed that cross-infection of AcSBV and AmSBV in both A. cerana and A. mellifera seems common, raising a concern of cross-infection intensifying the risk of disease resurgence in A. cerana. In this study, we analyzed the dynamics of cross-infection in three different types of apiaries (A. mellifera-only, A. cerana-only and two species co-cultured apiaries) over one year in Taiwan. Using novel, genotype-specific primer sets, we showed that SBV infection status varies across apiaries: AmSBV-AM and AcSBV-AC were the major genotype in the A. mellifera-only and the A. cerana-only apiaries, respectively, while AmSBV-AC and AcSBV-AC were the dominant genotypes in the co-cultured apiaries. Interestingly, co-cultured apiaries were among the only apiary type that harbored all variants and dual infections (i.e., AC and AM genotype co-infection in a single sample), indicating the interactions between hosts may form a conduit for cross-infection. The cross-infection between the two honey bee species appears to occur in a regular cycle with temporal fluctuation of AmSBV-AC and AcSBV-AC prevalence synchronized to each other in the co-cultured apiaries. Artificial infection of AcSBV in A. mellifera workers showed the suppression of viral replication, suggesting the potential of A. mellifera serving as a AcSBV reservoir that may contribute to virus spillover. Furthermore, the survival rate of A. cerana larvae was significantly reduced after artificial infections of both SBVs, indicating fitness costs of cross-infection on A. cerana and thus a high risk of disease resurgence in co-cultured apiaries. Our field and laboratory data provide baseline information that facilitates understanding of the risk of SBV cross-infection, and highlights the urgent need of SBV monitoring in co-cultured apiaries.

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Genomic Sequencing and Comparison of Sacbrood Viruses from Apis cerana and Apis mellifera in Taiwan.

Sacbrood virus (SBV) was the first identified bee virus and shown to cause serious epizootic infections in the population of Apis cerana in Taiwan in 2015. Herein, the whole genome sequences of SBVs in A. cerana and A. mellifera were decoded and designated AcSBV-TW and AmSBV-TW, respectively. The whole genomes of AcSBV-TW and AmSBV-TW were 8776 and 8885 bp, respectively, and shared 90% identity. Each viral genome encoded a polyprotein, which consisted of 2841 aa in AcSBV-TW and 2859 aa in AmSBV-TW, and these sequences shared 95% identity. Compared to 54 other SBVs, the structural protein and protease regions showed high variation, while the helicase was the most highly conserved region among SBVs. Moreover, a 17-amino-acid deletion was found in viral protein 1 (VP1) region of AcSBV-TW compared to AmSBV-TW. The phylogenetic analysis based on the polyprotein sequences and partial VP1 region indicated that AcSBV-TW was grouped into the SBV clade with the AC-genotype (17-aa deletion) and was closely related to AmSBV-SDLY and CSBV-FZ, while AmSBV-TW was grouped into the AM-genotype clade but branched independently from other AmSBVs, indicating that the divergent genomic characteristics of AmSBV-TW might be a consequence of geographic distance driving evolution, and AcSBV-TW was closely related to CSBV-FZ, which originated from China. This 17-amino-acid deletion could be found in either AcSBV or AmSBV in Taiwan, indicating cross-infection between the two viruses. Our data revealed geographic and host specificities between SBVs. The amino acid difference in the VP1 region might serve as a molecular marker for describing SBV cross-infection.

Comparison of gut microbiota of healthy and diseased walking sticks, Phasmotaenia lanyuhensis.

Research on gut microbiota of phytophagous insects has shown to be important for the physiological functions of insect hosts; however, little is known about the changes in gut microbiota when they are suffering from environmental stress or pathogen infections. During rearing of Phasmotaenia lanyuhensis (Phasmatodea: Phasmatidae), sluggish locomotion was usually followed by the death of the insect with a symptom of melanization in the front part of the abdomen. Therefore, the abnormal individuals were initially classified into moribund, light- and serious-symptom based on the level of abnormal physiological circumstances and melanization. The gut microbiota of these samples were further investigated by 16S metagenomic sequencing and the differences in bacterial abundance and structure of bacterial community were analyzed. A decrease in microbiota diversity was observed in the diseased P. lanyuhensis, with the abundance of phyla Proteobacteria and Firmicute relatively higher compared to those without symptom. Interestingly, principal component analysis based on the bacterial richness was correlated to the level of melanization symptom in the diseased P. lanyuhensis, suggested the change in bacterial microbiota involved in this abnormal circumstance. However, the factor that caused the initial alternation of microbiota remains to be identified. Additionally, the lack of bacterial diversity (i.e., absence of Meiothermus and Nubsella spp.) in P. lanyuhensis might reduce the fitness for surviving. This report provided the comprehensive microbiota analysis for P. lanyuhensis and concluded that either the relative abundance or the bacterial diversity of microbiota in the insect digestive system may influence the physiological functions of phytophagous insects.

Dynamics of Apis cerana Sacbrood Virus (AcSBV) Prevalence in Apis cerana (Hymenoptera: Apidae) in Northern Taiwan and Demonstration of its Infection in Apis mellifera (Hymenoptera: Apidae).

Since 2016, Apis cerana sacbrood virus (AcSBV) has been recorded in Taiwan. It is epizootic in Apis cerana (Hymenoptera: Apidae) and causing serious loss of A. cerana. Herein, we performed a long-term survey of AcSBV prevalence in the populations of A. cerana in Northern Taiwan from January 2017 to July 2018. The surveillance of AcSBV prevalence in A. mellifera (Hymenoptera: Apidae) populations was starting and further confirmed by sequencing since April 2017; thus, these data were also included in this survey. In our survey, the average prevalence rates of AcSBV were 72 and 53% in A. cerana and A. mellifera, respectively, in 2017, which decreased to 45 and 27% in 2018. For the spatial analysis of AcSBV in two honey bee populations, Hsinchu showed the highest prevalence, followed by New Taipei, Yilan, Taipei, and Keelung, suggesting that AcSBV might have come from the southern part of Taiwan. Interestingly, the AcSBV prevalence rates from A. cerana and A. mellifera cocultured apiaries gradually synchronized. The result of phylogenetic analysis and comparison of the annual AcSBV prevalence in A. cerana-only, A. mellifera-only, and A. cerana/A. mellifera cocultured sample sites indicate cross-infection between A. cerana and A. mellifera; however, AcSBV may lose the advantage of virulence in A. mellifera. The evidence suggested that the transmission of AcSBV might occur among these two honey bee species in the field. Therefore, A. mellifera may serve as a guard species to monitor AcSBV in A. cerana, but the cross-infection still needs to be surveyed.

Characterization and functional assay of apsup (Lyxy105) from Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV).

The baculoviral anti-apoptotic genes, p35 and iap (inhibitor of apoptosis), play important roles in the initiation of viral infection. Recently, a new anti-apoptotic gene (apoptosis suppressor, apsup) was identified in Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV). An apsup homolog gene, Lyxy105 (ly-apsup), was also predicted in the Lymantria xylina multiple nucleopolyhedrovirus (LyxyMNPV) genome. In this study, we attempt to perform a gene expression analysis and a functional assay of ly-apsup to demonstrate its anti-apoptotic activity and identify the functional domain of this protein. The transcription of the ly-apsup gene region was detected from 12 h post-infection (hpi) and increased significantly after 24-72 hpi. Comparison of the putative amino acid sequences to those of 18 baculoviral homolog proteins showed high amino acid identity to the LdMNPV sequences. Moreover, five conserved protein domains (named as domains I-V) were found. Therefore, protein functional assays were conducted on full-length proteins and different truncation clones. The overexpression of each clone was confirmed by western blot analysis, and the data revealed that a cleavage of ~ 5 kDa at the N-terminal region of the full-length, domains I-IV (1-241) and I-III (1-178), proteins occurred. The results of the functional analysis showed that full-length Ly-apsup and Ly-apsup with domain I (1-70) could inhibit Drosophila-RPR protein (D-RPR)-induced and actinomycin D (ActD)-induced apoptoses. In addition, the domains I and I-II (1-126) regions showed higher anti-apoptotic activity than the other domains in both D-RPR-induced and ActD-induced cell apoptoses. In conclusion, domain I of Ly-apsup may play an important role in the anti-apoptotic activity of this protein; cleavage of the Ly-apsup N-terminus may lead to decreased anti-apoptotic activity.

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

The phytogestrogenic stilbenes, arachidin-1 and resveratrol, modulate regulatory T cell functions responsible for successful aging in aged ICR mice.

CD4+CD25+ regulatory T cells (Tregs) are recognized as a distinctive T helper cell population which controls immunosuppression during the maintenance of immunological self-tolerance and immunohomeostasis. Sex steroids modulate fundamental immune functions, including immune cell development, differentiation and polarization, and facilitate specific immunophysiological microenvironments, such as pregnancy. The supplementation of exogenous phytoestrogens is beneficial to post-menopausal women. Stilbenes are a potent group of phytoestrogens, of which resveratrol (Res) is a well-known representative exhibiting a variety of immunomodulatory activities, including the attenuation of autoimmune diseases and boosting anti-tumor immunity. In the present study, arachidin-1 (Ara­1) and Res, primary stilbenes, enriched in peanut sprouts as phytoalexins, were investigated for their immunomodulatory properties for successful aging. We found that similar to 17-ß-estradiol (E2), Ara­1 or Res significantly inhibited concanavalin A (ConA)-activated lymphoblastogenesis of cell repertories from splenic or thymic origins. However, these inhibitory effects were partially reversed by the E2 receptor blocker, tamoxifen. While the ratios of the CD4+CD25+ cell population of ConA-activated T cell repertories were not significantly altered, treatment with E2, Ara­1 or Res led to an increase in the number of cytotoxic T-lymphocyte associated protein 4 (CTLA-4; also known as CD152)-positive cells and in the gene expression levels of CTLA-4, Forkhead box P3 (FoxP3), interleukin (IL)-10 and transforming growth factor-ß (TGF-ß). When low (L-S-PNT) and high (H-S-PNT) levels of stilbene-enriched peanut sprout-fortified diets were provided ad libitum to 12­week-old ICR mice for 48 weeks, their circulating Treg populations were assessed following magnetic bead enrichment. The gene expression levels of CTLA-4 and TGF-ß were significantly (P<0.05) elevated, as assessed by semi-quantitative RT-PCR. The findings of the present study support the beneficial roles of the phytoestrogenic stilbenes, Res and Ara­1, in facilitating a successful aging immune status which may attribute to longevity.

Modified fast procedure for the detection and screening of antiglycative phytochemicals.

In an attempt to elevate temperature to facilitate glycation, a nonenzymatic reaction by incubation of bovine serum albumin (BSA) and fructose at 50 °C for 24 h has been developed. As conducted and compared to a routine procedure by incubation of BSA and fructose at 37 °C for 168 h, the reactant fluorescence intensities and SDS-PAGE-detected glycated BSA quantities produced by both test temperatures increased with time of incubation. As the Amadori products and α-dicarbonyl compounds during incubation were quantified, both quantities produced at each temperature also increased with an increase of time of incubation, and their trends of changes at both temperatures were similar. In practical application for the detection and screening of the antiglycative phytochemicals, each of 20 peanut root extracts was introduced to a series of BSA-fructose solutions and incubated at 37 and 50 °C for 168 and 24 h, correspondingly. All extracts exhibited notable activities and varied depending on peanut origins. Pair comparison of the resultant antiglycative activities determined at 37 and 50 °C showed that both determined activities for each peanut root extract deviated limitedly. As further analyzed, SDS-PAGE-detected glycated BSA quantities formed at 50 °C were closely proportional to the antiglycative activities determined on the basis of their fluorescence intensities. It is of merit to demonstrate that fluorescence-based determination of BSA-fructose reactant after incubation at 50 °C for 24 h is practical and time-saving in the detection and screening of antiglycative phytochemicals.

Oral administration of Trapa taiwanensis Nakai fruit skin extracts conferring hepatoprotection from CCl4-caused injury.

As a folk medicine, the hot-water infusion of water caltrop fruits has been used to protect the liver. In this study, the outer skins of mature water caltrop fruits ( Trapa taiwanensis Nakai) were removed, forced-air-dried, pulverized, and subjected to extraction with hot water, and the infusion was lyophilized and pulverized to prepare a hot water extract of T. taiwanensis (HWETT). HWETT was subjected to assays of α,α-diphenyl-ß-picrylhydrazyl scavenging activity, reducing power, Trolox equivalent antioxidant capacity, and antioxidative potency, and all determinations showed HWETT to be a potent antioxidant. As further analyzed with LC-MS, two major HPLC-detected components were elucidated as gallic acid and ellagic acid. Hepatoprotective activity of HWETT was assessed with Sprague-Dawley male rats by oral administration. Six groups of rats (n = 8 for each) were respectively treated, namely, control, CCl(4) (20% CCl(4)/olive oil by 2.0 mL/kg bw), CCl(4) and Silymarin (200 mg/kg bw), CCl(4) and low HWETT dose (12.5 mg/kg bw), CCl(4) and medium HWETT dose (25 mg/kg bw), and CCl(4) and high HWETT dose (125 mg/kg bw). After 8 weeks, all animals were fasted for an additional day and sacrificed to collect blood, liver, and kidney for analyses. Histopathological examinations showed that oral administrations with Silymarin and HWETT were effective in protecting the liver from CCl(4)-caused fatty change. Oral administration of HWETT at 125 mg/kg bw was more effective than was Silymarin at 200 mg/kg bw. On biochemical analyses, oral administrations with HWETT at medium and high doses were effective (p < 0.05) in lowering CCl(4)-caused increases of alanine aminotransferase and aspartate aminotransferase activities. It is of merit to demonstrate HWETT as a potent source of antioxidants and hepatoprotective agents.

Oral administration of resveratrol in suppression of pulmonary metastasis of BALB/c mice challenged with CT26 colorectal adenocarcinoma cells.

Anti-cancer activities of resveratrol (3,4',5-trihydroxylstilbene) have attracted extensive research attention. Suppression of pulmonary metastasis of BALB/c mice challenged with CT26 colorectal adenocarcinoma cells achieved by oral administration of resveratrol was assessed in three separate experiments. Each mouse was challenged by tail vein injection with CT26 cells. Prior to challenge, 8-wk-old mice were fed with a basal diet and orally administered with resveratrol (30 mg/kg/2 days) eight or twelve times. After challenge, oral administration of resveratrol was continued until mice were sacrificed on day 20. As integrated from three experiments, 3.7% of the control mice (n=27) and 68.7% of the resveratrol-treated mice (n=26) exhibited free of metastasis. In a second study, 8-wk-old BALB/c mice were orally administered with resveratrol 12 times and challenged with CT26 cells for 100 days. All control mice died but 50% of the resveratrol-treated mice survived. The surviving mice were challenged with CT26 cells by hypodermic injection, fed with a basal diet for an additional 30 days, and sacrificed. Tumor lumps or nodules were not detected at the injection sites or in the lungs. This reveals that intrinsic vaccination-like defense has resulted from administration of resveratrol and challenge of tumor cells.

Solvent effects on extraction and HPLC analysis of soybean isoflavones and variations of isoflavone compositions as affected by crop season.

Spring (February to June) and fall (August to December) crops of soybean grown yearly in Taiwan with reverse temperature patterns provide a novel model to assess the effect of the crop season. In this study, three soybean cultivars, namely CH 1, VS-KS 2, and HBS, were grown for 2001 fall, 2002 spring, 2003 fall, 2004 spring, 2004 fall, and 2005 spring crops. The harvested and sun-dried soybeans were lyophilized, pulverized, and stored at -25 degrees C until HPLC analyses of isoflavone compositions were performed. As affected by extraction solvent and HPLC mobile phase, the amount of isoflavones extracted by methanol-H(2)O was higher than those extracted by acetic acid-acetonitrile. In addition, when both extracts were subjected to HPLC analysis with reversed C18 column run respectively with methanol-H(2)O and acetic acid-acetonitrile mobile phases, malonyldaidzin, malonylglycitin, and malonylgenistin were not detected in the former phase. Accordingly, all harvested soybeans were subjected to methanol-H(2)O extraction and HPLC analysis with the acetic acid-acetonitrile mobile phase. Among the detected soybeans, daidzin, genistin, malonyldaidzin, and malonylgenistin were the majors and glycitin, malonylglycitin, daidzein, and genistein were the minors of isoflavones. As affected by crop season for each cultivar grown for 3 years, daidzin, genistin, malonyldaidzin, and malonylgenistin contents of soybeans of the fall crops were significantly higher than those of their spring crops ( p < 0.05).

Biosynthesis enhancement and antioxidant and anti-inflammatory activities of peanut (Arachis hypogaea L.) arachidin-1, arachidin-3, and isopentadienylresveratrol.

UNLABELLED: Peanut is a potent plant to be induced to synthesize bioactive stilbenoids. Bioactivities of those stilbenoids except resveratrol have been meagerly investigated. When peanut kernels (Tainan 14, a Spanish cultivar) were imbibed, incubated 3 days for germination, sliced, incubated with artificial aeration, periodically sampled, lyophilized, extracted with methanol, and subjected to reverse-phase HPLC analysis, four major fractionations were detected and identified as trans-resveratrol (Res), trans-arachidin-1 (Ara-1), trans-arachidin-3 (Ara-3), and trans-isopentadienylresveratrol (IPD). During incubation of the peanut slices, contents of Res, Ara-1, and Ara-3 increased tremendously from initially trace or not detectable amounts up to 147.3, 495.7, and 2414.8 microg/g, corresponding to 20, 16, and 24 h of incubation, while IPD contents continued to increase up to 28 h (4474.4 microg/g). When the four stilbenoids and butylated hydroxytoluene (BHT) were subjected to antioxidant characterization by various measures, all have exhibited varied potencies of antioxidant activity. In particular, retardation of absorbance increase at 234 nm as formation of the conjugated diene hydroperoxides in a real pork oil system stored at 60 degrees C, supplement of Ara-1 at 100 microM has shown equivalent or even greater activity than did BHT. When the media were supplemented with Res, Ara-1, Ara-3, and IPD at 15 microM for cultivation of mouse macrophage RAW 264.7 cells activated by lipopolysaccharide (LPS), the LPS-induced extracellular production of prostaglandin E2 (PGE2) and nitric oxide (NO) was significantly inhibited by Ara-1 (p < 0.001), Res (p < 0.001), Ara-3 (p < 0.01), and IPD (p < 0.01). It is noteworthy and of merit that all test stilbenoids have exhibited potent antioxidant and anti-inflammatory activities and varied as affected by number of hydroxyl groups and isopentenyl or isopentadienyl moiety. KEYWORDS: Arachis hypogaea L.; peanut; groundnut; resveratrol; stilbenoids; arachidin; antioxidant; anti-inflammation.

Germination of peanut kernels to enhance resveratrol biosynthesis and prepare sprouts as a functional vegetable.

Bioactive benefits of resveratrol in the diets have attracted extensive interests of the public. Peanut is one of the potent natural sources of resveratrol. In this study, germination of peanut kernels to enhance resveratrol biosynthesis and preparation of sprouts as a functional vegetable was conducted. When the rehydrated kernels of three peanut cultivars were germinated at 25 degrees C and relative humidity 95% in dark for 9 days, resveratrol contents increased significantly from the range of 2.3 to 4.5 microg/g up to the range of 11.7 to 25.7 mug/g depending upon peanut cultivar. In comparison with the sprout components, resveratrol contents were highest in the cotyledons, slightly lower in the roots, and not detected in the stems. When the sprouts were heated in boiling water for 2 min, resveratrol contents varied in a limited range. Methanol extracts of the freeze-dried sprouts exhibited potent 1,1-diphenyl-2-picryl-hydrazyl scavenging activity and antioxidative potency against linoleic acid oxidation. These activities increased with an increase of germination time. After 9 days of germination, total free amino acid, sucrose, and glucose contents increased significantly while crude protein contents decreased and the large sodium dodecyl sulfate polyacrylamide gel electrophoresis protein molecules of the kernels were extensively degraded. From a practical viewpoint, it is of potency to prepare peanut sprouts as a functional vegetable.

Enhancement of tofu isoflavone recovery by pretreatment of soy milk with koji enzyme extract.

Isoflavones are novel nutraceutical constituents of soybeans, but considerable amounts are lost in the whey during conventional tofu manufacturing. In this study, in a small-scale process, 2 mL of koji enzyme extract (soybean koji/deionized water, 1/3, w/v) was combined with 600 mL of soy milk, and 30 mL aliquots were incubated at 35 degrees C for 0, 30, 60, 120, and 300 min, for enzyme pretreatment. After each treatment time, soy milk was heated to 85 degrees C, CaSO4 was added to aggregate protein, and the mixture was centrifuged to separate the solids (tofu) from the whey. The tofu yield and moisture contents from soy milk treated for 30 or 60 min were higher than those from soy milk treated for 0 (control), 120, or 300 min. The protein content of freeze-dried tofu varied in a limited range, and native PAGE and SDS-PAGE patterns revealed slight quantitative and qualitative variations among products. Soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased as the time of enzyme pretreatment of the soy milk increased. After 30 min of pretreatment, daidzin, genistin, daidzein, and genistein contents recovered in tofu products were higher than those of the control. In a pilot-scale process, aliquots (3 L) of soy milk were enzyme-treated for 30 min, aggregated with CaSO4, and hydraulically pressed to remove the whey. As in pretreatments, soy milk daidzein and genistein contents increased while daidzin and genistin contents decreased. In a comparison of the control and enzyme-treated tofu products, the total recoveries of daidzin, genistin, daidzein, and genistein in the tofu products increased from 54.9% to 64.2%. When the tofu products were subjected to a sensory panel test, both products were judged acceptable.