Changes in Chemical Compositions and Antioxidant Activities from Fresh to Fermented Red Mountain-Cultivated Ginseng

This study investigated changes in nutrients (fatty acids, amino acids, and minerals), ginsenosides, and volatile flavors, and antioxidant activities during food processing of mountain-cultivated ginseng (MCG) with the cocktail lactic acid bacteria. Fatty acid content increased, but the free amino acid content decreased, and minerals were practically unaffected during processing. Total phenolic and flavonoid contents and maillard reaction products increased markedly according to processing stage. The total ginsenosides levels increased from 31.25 mg/g (DMCG) to 32.36 mg/g (red MCG, RMCG) and then decreased (27.27 mg/g, at fermented RMCG) during processing. Particularly, the contents of F2 (0.31 → 1.02 → 2.27 mg/g), Rg3 (0.36 → 0.77 → 1.93 mg/g), and compound K (0.5 → 1.68 → 4.13 mg/g) of ginsenosides and β-panasinsene (17.28 → 22.69 → 31.61%), biocycloelemene (0.11 → 0.84 → 0.92%), δ-cadinene (0.39 → 0.5 → 0.94%), and alloaromadendrene (1.64 → 1.39 → 2.6%) of volatile flavor compounds increased during processing, along with to the antioxidant effects (such as DPPH, ABTS, and hydroxyl radical scavenging activities, and FRAP). This study may provide several choices for the use of ginseng in functional foods and functional cosmetics.     

Fermentation of Jamaican Cherries Juice Using Lactobacillus plantarum Elevates Antioxidant Potential and Inhibitory Activity against Type II Diabetes-Related Enzymes

Jamaican cherry (Muntinga calabura Linn.) is tropical tree that is known to produce edible fruit with high nutritional and antioxidant properties. However, its use as functional food is still limited. Previous studies suggest that fermentation with probiotic bacteria could enhance the functional properties of non-dairy products, such as juices. In this study, we analyze the metabolite composition and activity of Jamaican cherry juice following fermentation with Lactobacillus plantarum FNCC 0027 in various substrate compositions. The metabolite profile after fermentation was analyzed using UPLC-HRMS-MS and several bioactive compounds were detected in the substrate following fermentation, including gallic acid, dihydrokaempferol, and 5,7-dihydroxyflavone. We also found that total phenolic content, antioxidant activities, and inhibition of diabetic-related enzymes were enhanced after fermentation using L. plantarum. The significance of its elevation depends on the substrate composition. Overall, our findings suggest that fermentation with L. plantarum FNCC 0027 can improve the functional activities of Jamaican cherry juice.     

Alleviation of Ulcerative Colitis Potentially through th1/th2 Cytokine Balance by a Mixture of Rg3-enriched Korean Red Ginseng Extract and Persicaria tinctoria

Ginseng is a vastly used herbal supplement in Southeast Asian countries. Red ginseng extract enriched with Rg3 (Rg3-RGE) is a formula that has been extensively studied owing to its various biological properties. Persicaria tinctoria (PT), belonging to the Polygonaceae family, has also been reported for its anti-inflammatory properties. Ulcerative colitis (UC) is inflammation of the large intestine, particularly in the colon. This disease is increasingly common and has high probability of relapse. We investigated, separately and in combination, the effects of Rg3-RGE and PT using murine exemplary of UC induced by DSS (Dextran Sulfate Sodium). For in vitro and in vivo experiments, nitric oxide assay, qRT-Polymerase Chain Reaction (PCR), Western blot, ulcerative colitis introduced by DSS, Enzyme Linked Immunosorbent Assay (ELISA), and flow cytometry analysis were performed. The results obtained demonstrate that treatment with Rg3-RGE + PT showed synergism to suppress inflammation (in vitro) in RAW 264.7 cells via mitogen-activated protein kinase and nuclear factor κB pathways. Moreover, in C57BL/6 mice, this mixture exhibits strong anti-inflammatory effects in restoring colon length, histopathological damage, pro-inflammatory mediators, and cytokines amount, and decreasing levels of NLRP3 inflammasome (in vivo). Our results recommend that this mixture can be used for the prevention of UC as a prophylactic/therapeutic supplement.     

Evaluation and Functional Characterization of a Biosurfactant Produced by Lactobacillus plantarum CFR 2194

The study details the investigations on the ability of Lactobacillus plantarum CFR 2194, an isolate from kanjika, a rice-based ayurvedic fermented product, to produce biosurfactant. Surfactant production, as a function of fermentation time, indicates that the maximum production occurred at 72 h under stationary conditions. Isolation, partial purification, and characterization of the biosurfactant produced have been carried out, and Fourier transform infrared spectroscopy (FTIR) spectra demonstrated that biosurfactants were constituted by protein and polysaccharide fractions, i.e., possessed the structure typical of glycoprotein, which is affected by the medium composition and the phase of growth of the biosurfactant-synthesizing strain. Critical micelle concentration (cmc) of the biosurfactant was found to be 6 g l^?1. The emulsification index (EI), emulsification activity (EA), and emulsion stability (ES) values of the biosurfactant have confirmed its emulsification property. Aqueous fractions of the produced biosurfactant exhibited a significant antimicrobial activity against the food-borne pathogenic species: Escherichia coli ATCC 31705, E. coli MTCC 108, Salmonella typhi, Yersinia enterocolitica MTCC 859, and Staphylococcus aureus F 722. More importantly, the biosurfactant from L. plantarum showed antiadhesive property against above food-borne pathogens. The results thus indicate the potential for developing strategies to prevent microbial colonization of food contact surfaces and health-care prosthesis using these biosurfactants.     

Cell-Free Supernatants Obtained from Fermentation of Cheese Whey Hydrolyzates and Phenylpyruvic Acid by Lactobacillus plantarum as a Source of Antimicrobial Compounds, Bacteriocins, and Natural Aromas

Cheese whey hydrolyzates supplemented with phenylpyruvic acid (PPA) and commercial nutrients can be efficiently metabolized by Lactobacillus plantarum CECT-221 to biosynthesize some compounds with attractive applications in the food market. The main metabolites of cell-free extracts were antimicrobial compounds such as phenyllactic acid (PLA) and lactic acid (LA). The production of PLA by L. plantarum CECT-221 was evaluated in the Man–Rogosa–Sharpe broth supplemented with two biosynthetic precursors: phenylalanine or PPA. Using 30.5 mM PPA, the microorganism increased sevenfold the concentration of PLA producing 16.4 mM PLA in 46 h. A concentration of 40 mM PPA was a threshold to avoid substrate inhibition. The biosynthesis of whey hydrolyzates as a carbon source was enhanced by fed-batch fermentation of PPA; the average productivity of PLA increased up to 45.4?±?3.02 mM after 120 h with a product yield of 0.244 mM mM^?1; meanwhile, LA reached 26.1?±?1.3 g L^?1 with a product yield of 0.72 g g^?1. Cell-free fed-batch extracts charged in wells showed bacteriocin activity with halos of 7.49?±?1.44 mm in plates inoculated with Carnobacterium piscicola and antimicrobial activity against Staphylococcus aureus (11.54?±?1.14 mm), Pseudomonas aeruginosa (10.17?±?2.46 mm), Listeria monocytogenes (7.75?±?1.31 mm), and Salmonella enterica (3.60?±?1.52 mm). Additionally, the analysis of the volatile composition of the headspace of this cell-free extract revealed that L. plantarum is a potential producer for natural aromas, such as acetophenone, with high price in the market. This is the first report of PLA production from cheese whey and PPA. The extracts showed bacteriocin activity and potential to be applied as an antimicrobial in the elaboration of safer foods.     

Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH?=?7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30?°C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203.     

Plantaricin LD1: A Bacteriocin Produced by Food Isolate of Lactobacillus plantarum LD1

Plantaricin LD1, a bacteriocin produced by Lactobacillus plantarum LD1, was characterized for biochemical and antimicrobial properties. Bacteriocin showed stability at high temperatures (100 °C for 20 min and 121 °C for 15 min under 15 psi pressure), in a pH range of 2.0–8.0 and also in the presence of organic solvents, surfactants and detergents. The crude preparation was not affected by catalase, amylase and lipase but activity was reduced in the presence of pepsin, trypsin and proteinase K showing proteinaceous nature of the compound. The molecular weight of bacteriocin was found to be ～6.5 kDa, and antimicrobial activity was confirmed by bioassay. It inhibited not only related strains but also other Gram-positive and Gram-negative bacteria such as Lactobacillus curvatus NRRL B-4562, Lactococcus lactis subsp. lactis NRRL B-1821, Enterococcus faecium NRRL B-2354, Enterobacter cloacae NRRL B-14298, Micrococcus luteus, Staphylococcus aureus, urogenic Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri and Vibrio sp. These properties of plantaricin LD1 suggest its applications not only in food safety but in therapeutics as well.     

By-Product of the Red Ginseng Manufacturing Process as Potential Material for Use as Cosmetics: Chemical Profiling and In Vitro Antioxidant and Whitening Activities

Red ginseng (RG), which is obtained from heated Panax ginseng and is produced by steaming followed by drying, is a valuable herb in Asian countries. Steamed ginseng dew (SGD) is a by-product produced in processing red ginseng. In the present study, phytochemical profiling of extracts of red ginseng and steamed ginseng dew was carried out using gas chromatography-mass spectrometry (GC-MS) and rapid resolution liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry (RRLC-Q-TOF-MS) analysis. Additionally, antioxidant activities (DPPH, ·OH, and ABTS scavenging ability) and whitening activities (tyrosinase and elastase inhibitory activity) were analyzed. Phytochemical profiling revealed the presence of 66 and 28 compounds that were non-saponin components in chloroform extracts of red ginseng and steamed ginseng dew (RG-CE and SGD-CE), respectively. Meanwhile, there were 20 ginsenosides identified in n-butanol extracts of red ginseng and steamed ginseng dew (RG-NBE and SGD-NBE). By comparing the different polar extracts of red ginseng and steamed ginseng dew, it was found that the ethyl acetate extract of red ginseng (RG-EAE) had the best antioxidant capacity and whitening effect, the water extract of steamed ginseng dew (SGD-WE) had stronger antioxidant capacity, and the SGD-NBE and SGD-CE had a better whitening effect. This study shows that RG and SGD have tremendous potential to be used in the cosmetic industries.     

Extraction and Purification of R-Phycoerythrin Alpha Subunit from the Marine Red Algae Pyropia Yezoensis and Its Biological Activities

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe or as a colorant in the food and cosmetic industries. In this study, phycoerythrin was extracted from the red algae Pyropia yezoensis and purified by ammonium sulfate precipitation and various chromatography methods. The purified phycoerythrin was analyzed by UV-visible and fluorescence spectroscopy. The isolated pigment had the typical spectrum of R-phycoerythrin, with a trimmer state with absorbance maxima at 497, 536, and 565 nm. It was further purified and identified by LC-MS/MS and Mascot search. It showed a 100% sequence similarity with the R-phycoerythrin alpha subunit of Pyropia yezoensis. The molecular mass was 17.97 kDa. The antioxidant activity of the purified R-phycoerythrin alpha subunit was analyzed. It showed significant antioxidant activity in ABTS and FRAP assays and had significant cytotoxicity against HepG2 cells.     

超高效液相色谱法测定脐橙中橘红2号和苏丹红

采用超高效液相色谱法同时测定脐橙中的橘红2号和苏丹红染料。样品经乙腈超声提取,氨基固相萃取小柱净化后,用Waters ACQUITY UPLC BEH C18色谱柱分离,以乙腈-水为流动相进行梯度洗脱,采用二极管阵列检测器检测,检测波长分别为478 nm和515 nm。5种染料的质量浓度在0.20~20 mg·L-1范围内呈线性,检出限(3S/N)在0.31~0.53μg·kg-1之间。加标回收率在87.8%~99.4%之间,测定值的相对标准偏差(n=5)在0.54%~3.1%之间。     

Effect of Korean Red Ginseng and Rg3 on Asian Sand Dust-Induced MUC5AC,MUC5B,and MUC8 Expression in Bronchial Epithelial Cells

Korean Red ginseng (KRG), commonly used in traditional medicine, has anti-inflammatory, anti- oxidative, and anti-tumorigenic properties. Asian sand dust (ASD) is known to aggravate upper and lower airway inflammatory responses. BEAS-2B cells were exposed to ASD with or without KRG or ginsenoside Rg3. Mucin 5AC (MUC5AC), MUC5B, and MUC8 mRNA and protein expression levels were determined using quantitative RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor kappa B (NF-κB), activator protein 1, and mitogen-activated protein kinase expression and activity were determined using western blot analysis. ASD induced MUC5AC, MUC5B, and MUC8 mRNA and protein expression in BEAS-2B cells, which was significantly inhibited by KRG and Rg3. Although ASD-induced mucin expression was associated with NF-κB and p38 mitogen-activated protein kinase (MAPK) activity, KRG and Rg3 significantly suppressed only ASD-induced NF-κB expression and activity. KRG and Rg3 inhibited ASD-induced mucin gene expression and protein production from bronchial epithelial cells. These results suggest that KRG and Rg3 have potential for treating mucus-producing airway inflammatory diseases.     

气相色谱-离子阱串联质谱法测定人参中五氯硝基苯及其代谢产物残留

为了快速检测和确证人参中五氯硝基苯及其代谢物的残留量,建立了气相色谱/离子阱串联质谱法(GC-MS/MS)同时测定人参中五氯硝基苯(PCNB)及其2种代谢产物五氯苯胺(PCA)和甲基五氯苯硫醚(PC-TA)残留分析方法。样品用正己烷均质提取,弗洛里硅土固相萃取柱(Florisil-SPE)净化,采用GC-MS/MS多反应监测模式(MRM)检测,外标法定量。在人参中五氯硝基苯和五氯苯胺检出限均为1 ng/g,甲基五氯苯硫醚为0.1 ng/g;在0.01~0.5μg/g添加范围内,五氯硝基苯、五氯苯胺和甲基五氯苯硫醚的添加回收率分别为82.2%~89.4%、82.3%~84.7%和84.1%~88.5%;相对标准偏差均小于8.6%;检出限保持在0.1~1ng/g之间(信噪比10)。     

Survival of Freeze-dried Leuconostoc mesenteroides and Lactobacillus plantarum Related to Their Cellular Fatty Acids Composition during Storage

Lactic acid bacteria strains Lactobacillus plantarum CWBI-B534 and Leuconostoc ssp. mesenteroïdes (L. mesenteroïdes) Kenya MRog2 were produced in bioreactor, concentrated, with or without cryoprotectants. In general, viable population did not change significantly after freeze-drying (p?>?0.05). In most cases, viable population for cells added with cryoprotectants was significantly lower than those without (p?16:0), palmitoleic (C_16:1), stearic (C_18:0), oleic (C_18:1), linoleic (C_18:2), and linolenic (C_18:3) acids were identified. Four of them, C_16:0, C_16:1, C_18:0, and C_18:1, make up more than 94% or 93% of the fatty acids in L. mesenteroides and L. plantarum, respectively, with another one, namely, C18:3, making a smaller (on average 5–6%, respectively) contribution. The C_18:2 contributed very small percentages (on average?≤?1%) to the total in each strain. C_16:0 had the highest proportion at most points relative to other fatty acids. Moisture content and water activity (a _w) increased significantly during the storage period. It was observed that C_16:1/C_16:0, C_18:0/C_16:0 and C_18:1/C_16:0 ratios for freeze-dried L. mesenteroides or L. plantarum, with or without cryoprotectants, did not change significantly during the storage period. According to the packaging mode and storage temperatures, C_18:2/C_16:0 and C_18:3/C_16:0 ratios for freeze-dried L. mesenteroides and L. plantarum with or without cryoprotectants decreased as the storage time increased. However, a higher C_18:2/C_16:0 or C_18:3/C_16:0 ratio for L. mesenteroides and L. plantarum was noted in the freeze-dried powder held at 4 °C or under vacuum and in dark than at 20 °C or in the presence of oxygen and light.     

Characterization of a New Bacteriocin from Lactobacillus plantarum LE5 and LE27 Isolated from Ensiled Corn

Bacteriocins are low molecular peptides with antimicrobial activity, which are of great interest as food bio-preservatives and for treating diseases caused by pathogenic bacteria. In this study, we present the characterization of bacteriocins produced by Lactobacillus plantarum LE5 and LE27 isolated from ensiled corn. Bacteriocins were purified through ammonium sulfate precipitation and double dialysis by using 12- and 1-kDa membranes. Bacteriocins showed activity against Listeria innocua, Listeria monocytogenes, and Enteroccocus faecalis. Molecular weight was estimated through Tricine-SDS-PAGE and overloading the gel onto Mueller-Hinton agar seeded with L. monocytogenes, showing an inhibition zone between 5 and 10 kDa. NanoLC-MS/MS analysis allowed the identification of UPF0291 protein (UniProtKB/Swiss-Prot Q88VI7), which is also presented in other lactic acid bacteria without assigned function. Ab initio modeling showed it has an α-helix-rich structure and a large positive-charged region. Bacteriocins were stable between 4 and 121 °C and pH 2 and 12, and the activity was inhibited by SDS and proteases. Mode of action assay suggests that the bacteriocin causes of target microorganism. Taken together, these results describe a possible new class IIa bacteriocin produced by L. plantarum, which has a wide stability to physicochemical conditions, and that could be used as an alternative for the control of foodborne diseases.     

Use of Microwave Maceration in Red Winemaking: Effect on Fermentation and Chemical Composition of Red Wines

The objective of this study was to evaluate the effect of microwave treatment of crushed grapes on the yeast population of the must and on the development of alcoholic fermentation, as well as on the extraction of different compounds from the grapes such as polysaccharides and amino acids that can affect the organoleptic quality and stability of the wine. This study demonstrated for the first time the effect of the microwave treatment of grapes on native yeast species and their diversity, producing an increase in fermentation kinetics and a decrease in the lag phase. The microwave treatment produced a positive effect on the extraction of amino acids and polysaccharides from the grapes, resulting in significantly higher amounts of the main amino acids of the must and some major volatile compounds in the treated samples. The polysaccharides most affected by the microwave treatment were the PRAGs, the main polysaccharides liberated from grapes during the maceration.     

Effects of Fermentation and Storage on Bioactive Activities in Milks and Yoghurts

Fermentation of milk enhances its nutritional value through improved bioavailability of nutrients and production of bioactive substances which have biological functions. The goals of this research were to study the effect of fermentation and storage on antioxidant and antimicrobial activities in buffalo, goat and cow milks and yoghurts. Samples of buffalo, goat, cow milks and their yoghurts during their fermentation and storage were determined for proximate analysis and bioactive activities including antioxidant activities of DPPH, ABTS and reducing power assays, and antimicrobial activities against Staphylococcus aureus, Bacillus cereus, Salmonella typhimurium and Escherichia coli. Results showed that buffalo, cow and goat yoghurts had antioxidant activities in all assays and their activities significantly increased during their fermentation. Pasteurization did not affect the antioxidant activities. The activities of all yoghurts remained unchanged after a storage time of 21 days at 4 °C. For the antimicrobial activities, only yoghurts from buffalo, cow and goat milks had the activities, while all milks did not show any activities. However, buffalo yoghurt could inhibit only Gram positive strains (S. aureus and B. cereus), while goat and cow yoghurt inhibited all tested strains. A chemical responsible for antimicrobial activities in yoghurts was lactic acid formed by lactic acid bacteria. However, bioactive peptides produced by protein digestion during milk fermentation by lactic acid bacteria could not be ruled out for antioxidant and antimicrobial activities present. The antimicrobial activities of all yoghurts remained constant during their storage. It is concluded that all yoghurts would retain milk nutrition and bioactive functions during their storage in a refrigerator and may be served as a functional food with benefits from those activities for consumers.     

Antioxidant and Antimicrobial Activities of Erodium arborescens Aerial Part Extracts and Characterization by LC-HESI-MS2 of Its Acetone Extract

The phytochemical analysis of antioxidant and antibacterial activities of Erodium arborescens aerial part extracts constitute the focus of this research. The chemical composition of an acetone extract was investigated using LC-HESI-MS², which revealed the presence of 70 compounds. The major identified components were tannin derivatives. Total polyphenol and total flavonoid contents were assessed in plant extracts (hexane, ethyl acetate, acetone and methanol). The results showed that the acetone extract exhibited the highest contents of polyphenols and flavonoids, 895.54 and 36.39 mg QE/g DE, respectively. Furthermore, when compared to other extracts, Erodium arborescens acetone extract was endowed with the highest antioxidant activity with 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and total antioxidant capacity (TAC) tests. In addition, the four extracts of Erodium arborescens showed variable degrees of antimicrobial activity against the tested strains, and the interesting activity was obtained with acetone and methanol extracts.     

Discoloration Investigations of Yellow Lantern Pepper Sauce (Capsicum chinense Jacq.) Fermented by Lactobacillus plantarum: Effect of Carotenoids and Physiochemical Indices

Color is one of the important indicators affecting the quality of fermented pepper sauces, and it is closely related to carotenoid composition. This study systematically analyzed the changes in carotenoids and related physiochemical indices during the fermentation of yellow lantern pepper sauce. The CIELab color values indicated that L* and C* displayed a significant decreasing trend during fermentation. After 35 days of fermentation, the total carotenoid content significantly reduced from 3446.36 to 1556.50 μg/g DW (p < 0.05), and the degradation rate was 54.84%. Among them, the total content of carotene decreased by 56.03% during fermentation, whereas the degradation rate of xanthophylls and their esters was 44.47%. According to correlation analysis, violaxanthin myristate and lutein played a pivotal role in L*, a *, b *, chroma (C*), and yellowness index (YI). Moreover, PCA analysis indicated that lactic acid and acetic acid were the important qualities affecting the stability of pigment in fermented yellow lantern pepper sauce, which might also be the inducement of the color change. This work gives additional information concerning the discoloration of yellow lantern pepper sauce during fermentation and provides theory evidence regulating and improving the sensory qualities of yellow lantern pepper sauce.     

甲酚红分光光度法测定替米沙坦的含量

在pH为6.60的B-R缓冲介质中,替米沙坦(TMST)与甲酚红反应生成离子缔合物。当以试剂空白作参比时,在572nm及428nm波长处分别有正、负吸收峰。测定在572nm处增色反应的吸光度或在428nm处褪色反应的吸光度均与TMST的浓度之间呈线性关系,可作为测定TMST的基础。在所提出的方法中采用572nm作为测定TMST的检测波长。在此条件下TMST的浓度在4.6×10-7～1.3×10-5 mol.L-1之间与所测吸光度呈线性关系,其表观摩尔吸光率为5.34×104L.mol-1.cm-1。应用此方法测定了TMST胶囊和片剂中TMST的含量,测定结果与标示量相符,测定值的相对标准偏差(n=11)均小于2.5%。