Non-volatile organic acids (NVOAs) are essential to the flavor profile of Baijiu. However, the low levels and diversity of NVOAs in Baijiu make their isolation, annotation, and quantification challenging. In this study, a well-established pipeline combining chemical derivatization, isotope labeling, and high-resolution mass spectrometry with a three-tier annotation process was used to quantify NVOAs in three typical flavor types of Baijiu with high coverage and confidence. The results revealed the annotation of 56, 145, and 1277 NVOAs in Baijiu at tier 1, tier 2, and tier 3 levels, respectively. Among them, a total of 166 high-confidence NVOAs were first reported in Baijiu. Furthermore, multivariate statistical analyses indicated that abundant NVOAs could potentially be used as biomarkers to distinguish between different flavor types of Baijiu. This study provides a powerful tool for the qualification and quantification of NVOAs in Baijiu. The results will greatly expand the understanding of NVOAs in Baijiu.

This research aimed to address the issue of aflatoxin B1 (AFB1) contamination, which posed severe health and economic consequences. This study involved exploring unique species resources in the Qinghai-Tibet Plateau, screening strains capable of degrading AFB1. UPLC-Q-Orbitrap HRMS and NMR were employed to examine the degradation process and identify the structure of the degradation products. Results showed that Bacillus amyloliquefaciens YUAD7, isolated from yak dung in the Qinghai-Tibet Plateau, removed 91.7% of AFB1 from TSB-AFB1 medium with an AFB1 concentration of 10 μg/mL (72 h, 37°C, pH 6.8) and over 85% of AFB1 from real food samples at 10 μg/g (72 h, 37°C), exhibiting strong AFB1 degradation activity. Bacillus amyloliquefaciens YUAD7’s extracellular secretions played a major role in AFB1 degradation mediated and could still degrade AFB1 by 43.16% after boiling for 20 min. Moreover, B. amyloliquefaciens YUAD7 demonstrated the capability to decompose AFB1 through processes such as hydrogenation, enzyme modification, and the elimination of the -CO group, resulting in the formation of smaller non-toxic molecules. Identified products include C12H14O4, C5H12N2O2, C10H14O2, C4H12N2O, with a structure consisting of dimethoxyphenyl and enoic acid, dimethyl-amino and ethyl carbamate, polyunsaturated fatty acid, and aminomethyl. The results indicated that B. amyloliquefaciens YUAD7 could be a potentially valuable strain for industrial-scale biodegradation of AFB1 and providing technical support and new perspectives for research on biodegradation products.

Trichosanthes kirilowii Maxim. (Cucurbitaceae), one of the Chinese herbal medicines, is an economically important crop in Anhui Province, China. In recent years, gummy stem blight disease, a major disease of cucurbits, was widespread in many T. kirilowii plantations. The initial symptoms on the naturally infected stems appeared as dark brown water-soaked lesions, and as the disease progressed, vines of T. kirilowii gradually withered. On leaves, brown water-soaked lesions were visible initially, and then lesions enlarged and coalesced, resulting in extensive necrosis of leaves. On fruit, lesions covered with the white mycelium were nearly circular and tan to brown initially. Subsequently, the diseased fruit turned black and rotten commonly known as fruit rot or black rot. A Stagonosporopsis-like organism was consistently isolated from symptomatic stems, leaves and fruits. Fungal isolates were initially white and later turned dark grey or black with woolly to floccose aerial mycelium on PDA medium. Twenty-four isolates from different plantations were selected for further morphological studies. Pycnidia and conidia were formed after inoculating on cucumber fruit for 3 days. Pycnidia were globose to sub-globose, brown, ostiolate and 106.7 to 213.6 μm (average 160.1 μm, n = 50) in diameter. Conidia were hyaline, ellipsoidal, aseptate or one-septate, slightly constricted at the septa, 6.1 to 13.6 × 3.5 to 4.8 μm (average 9.9 × 4.1 μm, n = 50), and contained two or more oil drops. Three different loci of the genomic DNA, including the nuclear ribosome DNA internal transcribed spacer (ITS), RNA polymerase II second-largest subunit (RPB2), and β-tubulin (TUB2) genes., were amplified using primers ITS1/ITS4 (White et al. 1990), RBP2DF/RBP2DR (Lawrence et al. 2013), and T1/β-Sandy-R (O' Donnell and Cigelnik 1997; Stukenbrock et al. 2012), respectively and sequenced. A phylogenetic tree was built based on analysis of ITS, RPB2, and TUB2 sequences that deposited in GenBank (MW485497-MW485502 for ITS, MW531661-MW531666 for RPB2, and MW531667-MW531672 for TUB2), using the maximum likelihood method. The phylogenetic tree showed that the isolates fell into a single clade with S. cucurbitacearum. On the basis of morphological and molecular characteristics, the isolates obtained from T. kirilowii were identified as Stagonosporopsis cucurbitacearum. Pathogenicity tests were carried out on stems and leaves of 4-week-old T. kirilowii seedlings and on immature fruit collected from adult T. kirilowii plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were similarly inoculated with agar plugs. The diameters of lesions were measured in two perpendicular directions. Re-isolations from the stem and leaf lesions were performed on the PDA medium. Stagonosporopsis cucurbitacearum, was re-identified based on its colony and conidial characteristics and, therefore, completed Koch's postulates. Gummy stem blight caused by S. cucurbitacearum has been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on T. kirilowii caused by S. cucurbitacearum in China. The research provides a basis for the development and implementation of effective management strategies. Pathogenicity tests were carried out on stems and leaves of 4-week-old T. kirilowii seedlings and on immature fruits collected from adult T. kirilowii plants. The epidermis, previously injured with a syringe needle, was inoculated with 5-mm-diameter mycelial plugs, and the inoculated areas were then wrapped in water-soaked cotton. Controls were treated similarly but inoculated with agar plugs. Diameters of lesions were measured in two mutually perpendicular directions. Reisolations from the lesions were performed on PDA medium, and was re-identified based on its colony and conidial characteristics to complete Koch's postulates. Gummy stem blight caused by S. cucurbitacearum have been reported in a wide range of hosts, including cucumber, luffa, pumpkin, gourd, muskmelon, cantaloupe, and watermelon (Jiang et al. 2015; Keinath 2011; Zhao et al. 2019). To our knowledge, this is the first report of gummy Stem blight disease on T. kirilowii caused by S. cucurbitacearum in China. The research provides a basis for the development and implementation of effective management strategies.

Chinese cucumber, Trichosanthes kirilowii Maxim, is a perennial liana plant belonging to the Cucurbitaceae family and is an important traditional medicine in Chinese herbalism. The root, fruit and seed possess the medicinal value, and also the seeds are edible (Zhang et al. 2019). With increasing demand, the wild resource was domesticated and has been planted in China. Diseases of T. kirilowii have become more prominent with the expansion of cultivated area and have caused the yield reduction (Zhang et al. 2014). The general disease field has caused a yield reduction of 10% -30%, even up to 80% seriously. Since 2017, fields in Luan city, Anhui province exhibited 10 to 30% of plants with stem blight and fruit rot. The two-week seedlings were infected at the basal part of stem and showed water-soaking, then damping off. In older plants, it is common to see stem blight with brown-to-black lesions, stunted growth, and most diseased plants eventual death. On rot fruit, the symptom of water soaked lesions was firstly observed, and then with white mold, eventual rot. More than 60 samples of symptomatic stems or fruit were collected from Luan and Hefei, Anhui province during April to October 2017. The symptomatic tissues were washed, disinfested with 0.5% sodium hypochlorite for 30 s, rinsed twice in sterile water for 1 min, dried and incubated on V8-juice amended with 50μg ml-1 of ampicillin and rifampicin at 25°C. Based on morphological characteristics, more than 80% of the total 98 isolates generated were similar on the level of morphology, and preliminarily identified as Phytophthora species (Erwin and Ribeiro 1996). Three isolates were selected randomly to further observe and identify up to species level. The isolates produced abundant, aerial, white mycelia on V8 agar plates. Sporangia were produced on sporangiophores in 10% V8 liquid culture medium after 4 days at 25°C, mainly obovoid with one papilla, 38.8 to 50.4 μm× 22.8 to 33.5 μm in size. The single mature sporangium immersed in water quickly released 20 to 40 biflagellate motile zoospores. The isolates were further confirmed by amplification and sequencing of two conserved markers, the internal transcribed spacer (ITS) region and cytochrome c oxidase subunit I (COI) region with primers ITS1/ITS4 (White et al. 1990) and OomCoxILevup/Fm85mod (Robideau et al. 2011), respectively. The GenBank Accession Nos. were MN368092, and MN369544 for ITS and COI, respectively. The BLAST search results showed 100% similarity with ITS sequences (KF700090, KC438376) and COI sequences (MH136864, AY129166) of Phytophthora capsici isolates in Genbank. Koch's postulates were performed by testing the pathogenicity of the sequenced isolate on the Chinese cucumber (cv. 'Wanlou9'). On 1-month-old plants, a flap of bark was cut with a sterile scalpel, and the 2×2 cm plug of 5-day-old mycelium was inserted. The flap was then closed and sealed with Parafilm. The agar plug was treated in the same manner as the inoculated plants as the controls. And also, the intact fruit (one month old) was inoculated with 10 μL of zoospore suspension (2 × 105 zoospore/ml) and kept in growth chamber at 25 °C, with 80% relative humidity, and the control was treated with 10 μL of sterile distilled water. Three days after inoculation, the stems and the fruit inoculated with mycelium and zoospores showed water-soaked lesions. After 10 days, the symptoms on the tissues resembled those observed in the field. No symptoms were detected on the controls. P. capsici was reisolated from the diseased tissues but not from the control. This combination of data confirmed that the pathogen was P. capsici. To our knowledge, this is the first report of P. capsici causing stem blight and fruit rot on Chinese cucumber in China.

Trichosanthes kirilowii Maxim.seed oil extracted by Soxhletex traction were used as material.The acid value,iodine value and saponification value were analyzed.Trichosanthes kirilowii Maxim seed oil were esterified by acid catalyst,alkali catalyst and BF3catalyst.The differences in type and content between the different kinds of octadecatrienoic acid were studied via gas chromatography-mass spectrometry(GC-MS).The results showed that the acid value,the iodine value and the saponification value were 0.51 mg KOH/g,1.3398 g I2/g and 1.9143 g KOH/g.5 octadecatrienoic acids were identified by acid-catalyzed esterification method,being of punicic acid(6.43%),catalpa acid(9.5%),α-eleostearic acid(7%),γ-linolenic acid(5.82%),and α-linolenic acid(1.52%).4 octadecatrienoic acids were identified by alkali-catalyzed esterification method and BF3-catalyzed esterification method,being of punicic acid(31.24% and 27.17%),α-eleostearic acid(3.33% and 3.56%),catalpa acid(1.23% and 2.02%) and α-linolenic(0.38% and 0.78%).The quality indexes of the Trichosanthes kirilowii Maxim.seed oil met the national standards and it could be popularized.Alkali-catalyze esterification method was the most suitable method for Trichosanthes kirilowii Maxim.seed oil because of the minimum isomerization.

OBJECTIVE:To investigate the bacteria community and biodiversity of four-years pickled Yanshan Dongcai.METHODS:We studied the bacterial communities of Dongcai by 16S rDNA diversity analysis and the cultured species isolated from Dongcai sample by Restriction Fragment Length Polymorphism (RFLP) and 16S rRNA gene sequence analysis.RESULTS:The 16S rDNA diversity showed that the bacteria belonged to the phyla Proteobacteria (87.9% ) and Firmicutes (7.1% ), including many moderately halophilic bacteria such as Virgibacillus kekensis, Marinococcus albus, Salinicoccus sp., Lactobacillus halophilus and Halomonas. Only 5% of clone sequences belonged to the phylum Actinobacteria. Thirty-five strains were isolated from Dongcai sample, and 16S rDNA-RFLP analysis indicated that 34 isolates affiliated with the phylum Firmicutes, including Virgibacillus, Bacillus megaterium and Gracilibacillus saliphilus which were moderately halophilic bacteria, but only one isolate belonged to the phylum Actinobacteria.CONCLUSION:The bacterial diversity is low in Dongcai, dominated by moderately halophilic bacteria.

Phytosterol esters are kinds of novel functional food additives in lowering serum cholesterol and preventing cardiovascular disease.Phytosterol esters can be efficiently prepared from the sterols via lipase-catalyzed esterification with fatty acids and transesterification with fatty acid methyl esters or triacylglycerols.The synthesis methods of phytosterol esters via lipase-catalysis at home and abroad were described as well as development prospect,so as to offer reference for the research of phytosterol esters synthesis process in environmental protection,high productivity,feasibility,high security way.

Immobilized enzyme of Candida rugosa lipase on magnetic microsphere was used as biocatalyst for synthesis of phytosterol esters of alpha-linolienic.Response surface methodology(RSM) was employed to evaluate the effects of synthesis conditions.The optimum synthesis conditions were as follows: 25.2mg/mL immobilized CRL,146.5μmol/mL phytosterols concentration,at 55℃ for 5h in the presence of isooctane,the degree of phytosterols esterification was 92.61%.By solvent cleaning,the immobilization CRL sustained 78.1% activities after having been recycled 10 times.

This study is focused on the enzymatic esterification of phytosterols with different acyl donors to produce the corresponding phytosterol esters catalyzed by Canadia sp. 99-125 lipase under ultrasound irradiation. An ultrasonic frequency of 35kHz, power of 200W and time of 1h was determined to guarantee satisfactory degree of esterification and lipase activity. The influence of temperature, substrates concentration and molar ratio was investigated subsequently. The optimum production was achieved in isooctane system at 60°C with phytosterol concentration of 150μmol/mL and phytosterol to fatty acid molar ratio of 1:1.5, resulting in a phytosterol esters conversion of above 85.7% in short reaction time (8h). Phytosterols esters could also be converted in high yields to the corresponding long-chain acyl esters via transesterification with triacylglycerols (above 90.3%) under ultrasound irradiation. In optimum conditions, the overall esterification reaction rate using the ultrasonic pretreatment process was above 2-fold than that of mechanical stirring process without damage the lipase activity.

In this work, mixed-mode silica particles functionalized with octyl and sulfonic acid groups was conveniently prepared by co-bonding a mixture of n-octyltriethoxysilane and 3-mercaptopropyltriethoxysilane and then oxidized with hydrogen peroxide. Candida rugosa lipase (CRL) was immobilized on the mixed-mode silica particles via hydrophobic and strong cation-exchange interaction. The resulting immobilized CRL increased remarkably its stability at high temperature in comparison to free CRL. The immobilized CRL was used as biocatalysts for enzymatic esterification of phytosterols with free fatty acids (FFAs) to produce phytosterol esters. The phytosterols linolenate esterification degree of 95.3% was obtained under the optimized condition. Phytosterols esters could also been converted in high yields to the corresponding long-chain acyl esters via transesterification with methyl esters of fatty acids (80.5%) or triacylglycerols (above 95.5%) using mixed-mode silica particles immobilized CRL as biocatalyst. Furthermore, the immobilized CRL by absorption retained 78.6% of their initial activity after 7 recycles.

In this article, magnetic microspheres were prepared by suspension polymerization of allyl glycidyl and ethylene glycol dimethacrylate in the presence of vinylphosphonic acid-coated Fe3O4 nanoparticle. Candida rugosa lipase (CRL) was immobilized on the hydrophobic magnetic microspheres via the active epoxy groups. The resulting immobilized CRL had better resistance to pH and temperature inactivation in comparison to free CRL, the adaptive pH and temperature ranges of lipase were widened, and it exhibited good thermal stability and reusability. The immobilized CRL was used as biocatalyst for enzymatic esterification of phytosterols with unsaturated fatty acids (UFAs) to produce the corresponding phytosterol esters. The phytosterols linolenate esterification degree of 93.5% was obtained under the optimized condition: 100μmol/mL phytosterols, 200μmol/mL linolenic acid, 15mg/mL immobilized CRL at 160rpm and 55°C for 15h in 10mL of isooctane. Phytosterols esters could also be converted in high yields to the corresponding long-chain acyl esters via transesterification with methyl esters of fatty acids (55.3%) or triacylglycerols (above 78.1%) using magnetic immobilized CRL as biocatalyst.