Maianthemum atropurpureum (Franch) LaFrankie (Asparagaceae), called nibai in Tibetan or dongka in Drung or zhu-ye-cai in local Chinese, is a wild vegetable consumed by the Tibetan people and other ethnic groups in Northwest Yunnan, China. It is also a traditional medicinal plant used by different linguistic groups for antimicrobial purposes. However the nutritional and phytochemical compositions of this important medicinal food plant have not been well studied previously. In this study, the nutrient content for nibai was determined by the China National Standards (GB) methods, and the phytochemical analysis involved multiple chromatographic and spectral methods including LC-TOF-MS analysis. Dried nibai is a rich source of protein (ca. 24.6%), with 18 of the 21 common amino acids. The amino acid content of nibai can reach up to 17.9/100 g, with the essential amino acids as major contributors, corresponding to 42.3% of the total amino acids. Nibai contains rich mineral elements, dietary fiber, vitamins, β-carotene, carbohydrates, and lipids. The phytochemical content of nibai was examined by conventional isolation strategies, as well as HR-ESI-TOF-MS to detect and identify 16 compounds including nine steroid saponins and seven flavonoids. Among these compounds, uridine, adenosine, guanosine, and β-methyl-6-methyl-d-glucopyranoside were found from the genus Maianthemum for the first time. These results help to demonstrate that the local people’s practice of consuming Maianthemum atropurpureum is reasonable due to its high levels of vitamins, minerals, essential amino-acids, and phytochemicals. Nibai may be further developed in Tibet and surrounding regions, and beyond as a health food, nutraceutical, and/or dietary supplement product. Wild edible plants play an important role in furthering food security and improving the nutrition in the diets of people around the world, especially in poor rural communities . The utiThe ethnic groups including Nu, Dulong (Drung), Lisu, Yi, Pumi, Bai, Naxi and Tibetan as well in northwest Yunnan  of China, reside at high elevation areas with mountains and deep valleys. To better survive in these extreme conditions, local ethnic people often turn to wild (uncultivated) plants to supplement their diet and therebey enrich their food diversity. Ahmad and Pieroni found that certain wild edible plants have been embraced by a particular local culture because of their traditionally acquired knowledge-based principles, feelings, and manners . MoreoveMaianthemum Web. for food, including M. atropurpureum (Franch.) LaFrankie, M. purpurea  LaFrankie, M. oleracea (Baker) LaFrankie, M. tatsienense (Franch.) LaFrankie, M. forrestii (W.W. Sm.) LaFrankie, M. fuscum  LaFrankie, and M. henryi (Baker) LaFrankie  LaFrankie, called nibai in Tibetan, dongka in Drung, zhu-ye-cai in local language, or gao-da-lu-yao in Mandarin, a flavorful seasonal wild vegetable with unique flavors. It grows at high attitude on mountains, and can be collected in May when the snow has just melted (Maianthemum atropurpureum (nibai) were collected and directly consumed by the local people  of a group of seven species in aFrankie , belongit melted . It was l people . Now it Maianthemum atropurpureum is also considered to be an excellent medicine for annealing, heat-clearing, detoxicating, and lowering blood pressure in traditional Chinese medicine practice. The rhizome, as a folk medicine, has been used to treat lung ailment, rheumatism, menstrual disturbance, mammitis, cuts, bruises, kidney diseases, and also to activate blood circulation and to alleviate pain (Maianthemum atropurpureum (nibai) have been preliminarily investigated  LaFrankie were collected in Gongshan County, Nujiang Prefecture, NW Yunnan, China, in May 2013 and identified by Prof. Chun-Lin Long (Minzu University of China). The voucher specimen (No. 20130584) was deposited at the Herbarium of Minzu University of China. Some of the sample (300 g) was boiled with salt water and dried for the determination of nutrient content. The remaining sample (8.5 kg) was dried in the Sun directly for phytochemical studies.The aerial parts of 3), and dichloromethane (DCM) were of analytical grade and were purchased from Beijing Chemical Works . Chromatography and extraction solvents  were of HPLC grade obtained from VWR Inc . UPLC grade water was prepared by using the Millipore Milli-RO 12 system . All pure isolated compounds were characterized by HR-ESI-MS, 1H NMR, and 13C NMR spectroscopic methods for identification purposes.The general laboratory solvents, including methanol (MeOH), ethanol (EtOH), petroleum ether (Pet), ethyl acetate (EtOAc), n-butyl alcohol (n-BuOH), acetone, chloroform  method (GB 5009.3-2010) . Ash wasTotal fiber, soluble fiber, and insoluble fiber contents were determined by Dietary Fiber Determination System  using the GB/T 5009.88-2008 method .β-carotene were analyzed according to corresponding China standard methods GB/T (2 was determined using molecular fluorescence photometer (β-carotene was carried out by high-performance liquid chromatography (HPLC) as previously reported  . Data weMaianthemum atropurpureum (nibai) (8.5 kg) were extracted five times with 70% EtOH under reflux to give an ethanol extract. After evaporation under reduced pressure, the extracted residue was suspended in water and partitioned successively with petroleum ether, ethyl acetate, and n-BuOH. The n-BuOH extract (109 g) was subjected to silica gel (200–300 mesh) column chromatography eluting with EtOAc-acetone gradient (1:0→0:1) and pooled based on TLC profiles to yield seven fractions (Fr.1-Fr.7). Fr.2 (3.24 g) was dissolved with MeOH, and the insoluble part was recrystallized with MeOH and gave compound 1. The soluble part was subjected to silica gel (200–300 mesh) column chromatography eluting with CH2Cl2-MeOH gradient (25:1→2:1) to yield Fr.2.3 and Fr.2.5. Fr.2.3 was purified by Sephadex LH-20 chromatography using MeOH as the eluent to yield compound 3. In addition, Fr.2.5 was repeated chromatographed by silica gel column under different gradient conditions with CH2Cl2-MeOH to give compounds 4, 5, and 7. The insoluble part in Fr.3 (3.01 g) was also recrystallized with MeOH and gave compound 2, and the soluble part was further purified by successive silica gel column chromatography with CH2Cl2-MeOH  followed by CH2Cl2-MeOH , yielding 8 mg of compound 6. Fr.5 (4.47 g) was subjected to Si gel column chromatography with CH2Cl2-MeOH  and pooled based on TLC profiles to yield five fractions (Fr.5.1-Fr.5.5). Fr.5.3 was purified by silica gel column chromatography  to yield compound 10. Fr.5.4 was repeated silica gel medium pressure liquid chromatography gradient eluted with a MeOH-H2O solvent system , followed by preparative HPLC with 50% MeOH (MeOH-H2O) and further purified by silica gel column to yield compounds 8 and 9.Dried and powdered samples of LC-TOF-MS analysis of the n-BuOH extract was performed using a Waters  Alliance 2695 system equipped with 2695 separation module units and 2998 PDA detectors using a 100 mm × 2.0 mm, 2.5 µm Phenomenex Synergi Hydro-RP 100 A column with 3 mm × 4.0 mm Phenomenex Security Guard column . The mobile phase consisted of solvents (A) 0.1% aqueous formic acid solution and (B) acetonitrile using a stepwise gradient elution of 3% B for 5 min, 3%–15% B at 5–15 min, 15% B for 5 min, 15%–45% B at 21–35 min, 45%–90% B at 35–40 min, and this proportion of solvent kept for 10 min. The UV-Vis spectra were recorded from 190 to 700 nm. The flow rate and the injection volume were 0.2 ml/min and 10 μL, respectively. Both column and sample temperatures were 25°C. LC/MS-grade methanol was used to redissolve the freeze-dried sample, and then brought up to 2 mg/ml and filtered using 25 mm syringe filter (0.45 µm PTFE membrane) prior to injection.High-resolution electrospray ionization time of flight mass spectrometry (HR-ESI-TOF-MS) was performed using an LCT Premier XE TOF mass spectrometer  equipped with an ESI interface and controlled by Mass Lynx V4.1 software. Mass spectra were acquired in both positive and negative modes over range m/z 100–1500. The capillary voltages were set at 3000 V (positive mode) and 2700 V (negative mode), respectively, and the cone voltage was 30 V. Nitrogen gas was used for both the nebulizer and desolvation. The desolvation and cone gas flow rates were 600 and 20 L/h, respectively. The desolvation temperature was 400°C, and the source temperature was 120°C.Maianthemum atropurpureum (Franch.) LaFrankie (nibai) are listed in Pteridium aquilinum var. latiusculum) with 25.5/100 g, one of the most important wild food plants in China (Spinacia oleracea) with 12.7/100 g. The protein and carbohydrate content among three species are very different; nibai has the highest protein content (24.6/100 g), and the lowest carbohydrate content (23.8/100 g). The protein and carbohydrate content of nibai is much higher than different vegetable amaranth species, such as Amaranthus blitum , followed by aspartic acid (1.73 g/100 g), leucine (1.71 g/100 g), lysine (1.32 g/100 g), and alanine (1.29 g/100 g), respectively.The 18 amino acids identified and quantified in rst time . The totrst time . The comnibai is a good source of amino acids, which can provide indigenous people with nearly all the amino acids that humans require. Four amino acids in nibai, aspartic acid and glutamate contribute to its umami taste, while glycine and alanine contribute to its sweet taste  , uridine 2) (β-D-glucopyranoside 5) (β-methyl-6-methyl-d-glucopyranoside 6) (O-robinobioside 7) (Maianthemum for the first time.Seven known compounds  have beeidine 2) , adenosiidine 2) , 2-phenyoside 5) , β-methyoside 6) , and isooside 7) . Among tMaianthemum atropurpureum comparing with current literatures . The identification of uridine 2) which was isolated as colorless acicular crystal was confirmed by comparing the 13C NMR data with literature and database values  was white powder; 1H NMR and 13C NMR were in agreement with literature values. ESI-MS m/z 267 [M + H]+  corresponded with those reported in the literature . Guanosine 4) was identified as white powder; 1H NMR and 13C NMR were in agreement with the literature  and was identical to literature data (β-d-glucopyranoside 5) was gummy, colorless, solid; 1H NMR13C-NMR are in agreement with published values. ESI-MS m/z 285 [M + H]+  was identical to literature data (β-methyl-6-methyl-d-glucopyranoside 6) was white powder; 13C NMR : δ 103.5 (C-1), 78.2 (C-3), 74.1 (C-5), 73.7 (C-2), 72.5 (C-6), 71.9 (C-4), 66.5 (C6-OCH3), 64.5(C1-OCH3); ESI-MS m/z 208 [M] . Comparing with NMR database, the 13C NMR data showed 75% similarity to that of ethyl-β-D-fructopyranoside, from which the hydroxyl substituent fructose was confirmed. The 1H NMR spectrum of 6 showed the presence of six hydrogens at δ1.74 and 2 carbons at δ 66.5 and 64.5, pointing to the existence of two methoxy groups. Comparing all the carbon chemical shift data of β-methyl-d-glucopyranoside with 6-O-methyl-β-d-glucopyranoside, the data were in agreement with those obtained from the literature data (β-methyl-6-methyl-d-glucopyranoside. Isorhamnetin-3-O-robinobioside (7): pale yellow crystal; 1H NMR and 13C NMR are in agreement with published data  (Thymidine 1) was isolated as colorless acicular crystal. All NMR data are in agreement with literature data . The molecular formula was obtained e values . ESI-MS terature ; ESI-MS ure data . 2-Phenyure data . β-methyure data . Therefohed data , ESI-MS 16, 467) .nibai are attributed to its diverse bioactive components, especially the main constituents. According to this study and previous literature, the primary chemical components of M. atropurpureum are steroid saponins, nucleosides, and flavonoids, especially the large polarity constituents, some of which have considerable biological activities. For instance, the smilacinoside A, aspidistrin, and funkioside D isolated from the aerial parts of M. atropurpureum exhibited prominent activity in vitro cytotoxicity against K562 tumor cell line with IC50 values of 1.09, 0.47, and 2.93 μg/ml, respectively , an endemic species to the Hengduan Mountains, is a less common wild vegetable rich in nutrient and phytochemical content. It plays an important role not only in providing local Tibetans and other linguistic groups with various essential nutrition elements, but may also contribute to maintaining health, promoting immunity, and preventing several kinds of diseases. Consequently, nibai can be considered a promising new functional health food and/or nutraceutical.Many studies have correlated diet and certain chronic diseases such as cancer, cardiovascular disease, diabetes, and osteoporosis. Bioactive compounds from edible plants have the potential to prevent certain chronic conditions . Nibai,


A total of 94 patients, 44 men and 50 women, between 35 and 91 years old (mean ± SD: 63 ± 11.2), were enrolled. Fifty-two out of ninety-four participants underwent SiPM PET/CT first and a total of 162 lesions were detected using both scanners. Forty-two out of ninety-four participants underwent conventional PET/CT first and a total of 108 lesions were detected using both scanners. Regardless of whether SiPM-based PET/CT was used first or second, maximum standardized uptake value (SUVmax) of lesions measured on SiPM was on average 20% higher when comparing two scanners with all enrolled patients, and the difference was statistically significant. SiPM-based PET/CT detected 19 more lesions in 13 patients compared with conventional PET/CT. No lesions were only identified by conventional PET/CT. In conclusion, we observed higher SUVmax for lesions measured from SiPM PET/CT compared with conventional PET/CT regardless of the order of the scans. SiPM PET/CT allowed for identification of more lesions than conventional PET/CT. While delayed imaging can lead to higher SUVmax in cancer lesions, in the series of lesions identified when SiPM PET/CT was used first, this was not the case; therefore, the data suggest superior performance of the SiPM PET/CT scanner in visualizing and quantifying lesions.We prospectively enrolled patients with neuroendocrine tumors (NETs). They underwent a single  At the time of that study, the DMI PET/CT had not been approved by the Food and Drug Administration (FDA); therefore, the standard scanners were always used first, followed by SiPM-based PET/CT [Over the last decade, new photosensors, the silicon photomultipliers (SiPMs), were introduced into commercial PET systems . We prev imaging . At the 68Ga-DOTATATE to evaluate differences in quantitative and semi-quantitative measurements.Here we prospectively compared the DMI and D690 scanners in patients with NETs following a single injection of Patients with NETs were prospectively enrolled in this protocol between April 2017 and April 2019 and randomized to be scanned first using DMI followed by D690 or to be scanned first using D690 followed by DMI. The study was approved by the Stanford University institutional review board, and all participants signed an informed consent.The CT scan was obtained for attenuation correction and anatomical localization using 120 kV, “smart” modulating mA and a 512 × 512 matrix size. Thereafter, a whole-body (vertex to mid-thighs) PET scan was acquired in 3D mode with ToF enabled. D690  has an axial field of view of 15.7 cm. Each field of view contains 47 slices (3.27 mm), and the overlap between bed positions was set to 11 slices (23%). The acquisition time was 3 min per bed position. PET data were corrected using the segmented attenuation data of the CT scan and reconstructed using the vendor-recommended reconstruction protocol with an ordered subset expectation maximization (OSEM) with 2 iterations and 24 subsets. When performed as second scan, a low-dose CT (10 mA) was obtained for attenuation correction and anatomic localization to minimize radiation exposure to participants.The CT scan was obtained for attenuation correction and anatomical localization using 120 kV, “smart” modulating mA and a 512 × 512 matrix size. Thereafter, a whole-body (vertex to mid-thighs) PET scan was acquired in 3D mode with time-of-flight (ToF) enabled. DMI  has an axial field of view of 20 cm. Each field of view contains 71 slices (2.79 mm), and the overlap between bed positions was set to 17 slices (24%). The acquisition time was 3 min per bed position. PET data were corrected using the segmented attenuation data of the CT scan. Although reconstruction using the block sequential regularized expectation maximization (BSREM) protocol  was avaimax and SUVmean) images were displayed in a trans-axial view and a 3D region-of-interest (ROI) was placed in up to six detected lesions with the highest uptake. All lesions recorded on the first scan were matched to lesions on the second scan. We also recorded all the lesions detected only using SiPM scanner. A 2D circular ROI was used to measure uptake in normal organs . Location of ROIs for single background organs was decided by consensus between the two readers prior to the start of the analysis. Size of ROIs changed depending on the structure of interest. The ROIs for the liver were drawn excluding any lesions. The ROIs for the mediastinal blood pool were placed on the aortic arch. Contrast recovery (CR) was calculated as the ratio between lesional SUVmax and blood pool SUVmean. Measurements from both the SiPM and the conventional PET/CT were recorded from images reconstructed with OSEM and TOF.Scans were independently reviewed and analyzed by two nuclear medicine physicians  using Advantage Workstation . Any disagreements were resolved by consensus. Paired scans  were displayed using a fixed SUV scale (threshold 50%) and color table. For the evaluation of semiquantitative metrics  and STATA RELASE 14.2 . Continuous data are presented as mean ± standard deviation (SD), minimum-maximum values, and frequencies (%). Since the data did not follow a normal distribution, we performed the Wilcoxon signed ranked test to compare SUV68Ga-DOTA-TATE dosage ranged from 3.5 to 7.3 mCi (mean ± SD: 5.3 ± 0.8 mCi). The time from injection to the scan ranged from 42.9 to 93.5 min (mean ± SD: 61.3 ± 11) and from 62.5 to 143.6 min (mean ± SD: 88.1 ± 16.0) for the first and the second scan, respectively. The delay time between the two scans ranged from 17.6 to 61.8 min (mean ± SD: 26.8 ± 9.5). One hundred and eight patients with NETs were prospectively enrolled in this protocol between April 2017 and April 2019. However, 14 of the 108 patients were excluded after enrollment: 10 withdrew their consent and 4 decided to not undergo the second scan. p > 0.05). These results are shown in Agreement analysis showed that the two scanners were comparable in terms of normal background organs uptake, independent from the time delay (max ranged from 1.2 to 225 (mean ± SD: 31.4 ± 31.6) and from 0.9 to 234.3 (mean ± SD: 29.4 ± 29.5) for SiPM and conventional PET/CT, respectively. The difference in SUVmax measurements between the two scanners was statistically significant (p < 0.001). For semi-quantitative analysis, we measured up to 6 lesions with the highest uptake and identified by both scanners: a total of 270 NET lesions in 70 out of 94 (74%) patients were detected by both scanners. SUV68Ga-DOTA-TATE dosage ranged from 3.5 to 6.7 mCi (mean ± SD: 5.1 ± 0.8). Imaging started between 46.2 and 93.5 min (mean ± SD: 63.3 ± 10.7) and between 66.6 and 143.6 min (mean ± SD: 88.7 ± 16.7) after injection for SiPM and conventional PET/CT, respectively. The delay time between the two scans ranged between 17.6 and 61.8 min (mean ± SD: 25.4 ± 9.7). Fifty-two patients, twenty-three men and twenty-nine women, 43 to 85 years old (mean ± SD: 63.3 ± 10.4) underwent SiPM PET/CT first, followed by conventional PET/CT. BMI ranged from 18.2 to 44.8 (mean ± SD: 28.4 ± 5.8). max for these 162 lesions ranged from 3.2 to 118.8 (mean ± SD: 28.7 ± 19.6) and from 2.5 to 109.0 (mean ± SD: 27.6 ± 19) for SiPM and conventional PET/CT, respectively; the difference in SUVmax trended towards statistical significance (p = 0.049).A total of 162 lesions were detected in 38 patients  using both scanners. SUVmax was analyzed per single organ, the average measurements between the two scanners were significantly different for bone lesions (p = 0.03), trending towards significance for lymph nodes (p = 0.052) and not significant for liver, pancreas, and other sites of disease. Results are shown in When SUVp = 0.055). CR was on average higher for SiPM-based compared with conventional PET/CT and ranged from 3.5 to 127.1 (mean ± SD: 38.9 ± 32.1) and from 4.1 to 137.4 (mean ± SD: 35.4 ± 30), respectively; this difference was not statistically significant . These lesions had SUVmax measurements ranging from 1.7 to 13.3. (mean ± SD: 7.1 ± 4). There were no lesions seen only on conventional PET/CT.Compared with the conventional PET/CT, SiPM PET/CT identified 12 more An example is shown in 68Ga-DOTA-TATE dosage ranged from 3.6 to 7.3 mCi (mean ± SD: 5.5 ± 0.9 mCi). Imaging started between 42.9 and 90.7 min (mean ± SD: 58.8 ± 10.9) and between 62.5 and 138.4 min (mean ± SD: 87.4 ± 15.1) after injection for conventional and SiPM PET/CT, respectively. The delay time between the two scans ranged between 17.0 and 50.2 min (mean ± SD: 28.5 ± 9).Forty-two patients, twenty-one men and twenty-one women, between 35 and 91 years old (mean ± SD: 62.8 ± 12.2) underwent conventional PET/CT first, followed by SiPM PET/CT. BMI ranged from 17.6 to 39.8 (mean ± SD: 27.2 ± 5.5). max for these 108 lesions ranged from 0.9 to 234.3 (mean ± SD: 32 ± 40.4) and from 1.2 to 225 (mean ± SD: 35.4 ± 43.7) for conventional and SiPM PET/CT, respectively. This difference in SUVmax measurements was statistically significant (p < 0.001).A total of 108 lesions were detected in 32 patients  using both scanners. SUVmax difference (R2 = 0.18).Since in this sub-cohort of patients, the SiPM-based system was used for the second scan, we attempted to compare the two scanners after controlling for the time difference. A linear regression analysis was performed, and a weak association was found between acquisition time delay and the SUVmax measurements were analyzed per single organ lesions, the average values between the two scanners were significantly different for hepatic and pancreatic lesions  but not for lymph nodes, bone lesions, and other site of disease. Results are shown in When SUVp < 0.01).CR ranged from 1.3 to 262 (mean ± SD: 41.7 ± 59.2) and from 1 to 227.6 (mean ± SD: 44.8 ± 56.6) for conventional and SiPM-based PET/CT data, respectively. This difference was statistically significant . These lesions had SUVmax measurements ranging from 3.7 to 8.3 (mean ± SD: 5.6 ± 1.6). There were no lesions seen only on conventional PET/CT.Compared with the conventional PET/CT, SiPM PET/CT identified 7 more An example is shown in Characteristics of the patients who had lesions identified only using SiPM PET/CT are shown in max values were consistently higher for the SiPM-based system images than for conventional PET/CT. SiPM PET/CT detected 19 more lesions.Our results suggest better performance of SiPM PET/CT compared with conventional PET/CT regardless of the order of the scans. This was noted both in terms of overall detection and semi-quantitative measurements. Average SUV18F-FDG or 18F-Choline PET/CT and compared SiPM-based vs. conventional PET/CT scanners in terms of SUVmax of the target lesions and of mediastinal blood pool and liver as background organs. They reported that semi-quantitative values from the SiPM-based PET/CT were always higher than from the conventional ones, regardless the order of the scan; the difference was significant for detected lesions and blood pool but not for liver. In our study, we recorded higher SUVmax values when using SiPM-based PET/CT, both for the lesions analyzed and for single organs analysis. SUVmax was higher when SiPM PET/CT was performed as second scan independent from the time delay (R2 = 0.18), similar to what Ocampo et al. reported in their study (R2 = 0.29). We calculated CR, and the average values from SiPM-based PET/CT were higher. These findings may be explained by the better spatial resolution, photon sensitivity, and time-of-flight SNR gain for this SiPM-based PET system compared with the conventional PET/CT system used  [Other studies evaluated the difference between SiPM-based vs. conventional PET/CT scanners, reporting better performance of the SiPM-based systems ,12,13. F18F-FDG or 18F-Choline PET/CT between SiPM-based and conventional PET/CT. Their results indicate improved image quality when using SiPM-based PET/CT and more lesions detected by the SiPM-based PET/CT in 22 patients. Additionally, disease stage was modified in 7 out of those 22 patients based on findings from SiPM-based PET/CT. In our study, SiPM-based PET/CT identified 19 more lesions in 13 patients compared with conventional PET/CT, and most of them were lymph nodes metastases, which have important implications for treatment decisions and can be difficult to identify by conventional structural imaging [López-Mora et al.  compared imaging . Howevermax was reported. Comparison between background organs showed that no significant differences were registered between the two scanners, confirming the results in the previous study [We previously reported the higher performance of SiPM-based PET/CT in a cohort of participants who underwent FDG PET/CT for oncological indications . They weus study .®, GE Healthcare) was not used to reconstruct SiPM PET/CT images. BSREM controls noise at higher iterations, and we have shown that it improves image quality compared with the ordered subsets expectation maximization algorithm (OSEM) [This study has limitations: lesions detected only by the SiPM PET/CT scanner were not confirmed with biopsy, so some may have been false positive findings. Moreover, the block sequential regularized expectation maximization (BSREM) algorithm (Q.Clearm (OSEM) . Neverthmax measurements were higher in lesions detected by SiPM than by conventional PET/CT, regardless of the order of the scan. There was a better CR in images acquired using SiPM PET/CT. Nineteen lesions in thirteen patients were only identified using the SiPM PET/CT. The results indicate that SiPM PET/CT has superior performance compared with a conventional PET/CT scanner.The SUV


Salmonella Enteritidis, Listeria monocytogenes, and Escherichia coli). The results showed that minimum inhibitory concentrations (MICs) ranged between 0.031–1.0 mg/mL, although most bacterial groups (75%) showed MICs of 1.0 mg/mL. The reduction in the cell viability of pathogens due to ε-PL depended on the time and concentration, and 1/2 × MIC of ε-PL killed 99.99% of pathogens after 10 h of incubation. To confirm biofilm inhibition and degradation effects, crystal violet assay and confocal laser scanning microscopy (CLSM) were used. The biofilm formation rates of four bacterial groups  were 10.36%, 9.10%, 17.44%, and 21.37% at 1/2 × MIC of ε-PL, respectively. Additionally, when observed under a CLSM, ε-PL was found to induce biofilm destruction and bacterial cytotoxicity. These results demonstrated that ε-PL has the potential to be used as an antibiotic and antibiofilm material for chicken meat processing.ε-Polylysine (ε-PL) is a safe food additive that is used in the food industry globally. This study evaluated the antimicrobial and antibiofilm activity of antibacterial peptides (ε-PL) against food poisoning pathogens detected in chicken ( Salmonella, Campylobacter, enterohemorrhagic Escherichia coli, Listeria, and Vibrio cholerae are among the most common foodborne pathogens [Foodborne diseases commonly occur due to food being contaminated by bacteria, bacterial toxins, viruses, parasites, chemicals, and other agents. Researchers have identified more than 250 foodborne diseases . Common athogens .Campylobacter and occasionally by Clostridium perfringens and Salmonella [Listeria monocytogenes and E. coli have also been detected in cooked chicken [Globally, people enjoy eating poultry meat and its products. However, it is particularly important to pay attention to sanitation as chicken meat and its products can transfer foodborne pathogens . Raw chilmonella . Listeri chicken ,6. The g chicken . In addi chicken .Microorganisms normally live in complicated biological communities, known as biofilms, which provide higher resistance against external physical and chemical attacks such as high temperature, pH, humidity, bactericides, and other antibiotics . BiofilmMulti-species biofilms are biofilms composed of multiple microbial species. Each microbial species has its own characteristics and reveals some developed and distinct functions on multi-species biofilms that do not exist in their mono-species biofilms . Multi-sl-lysine, consisting of 25–35 residues [Streptomyces albulus and Lysinopolymerus, and can inhibit most bacteria, yeast, and viruses as AMPs [Antimicrobial peptides (AMPs) are oligopeptides that consist of five or more amino acids , and areresidues . It is p as AMPs . In addi as AMPs . HoweverSalmonella Enteritidis KCCM 12021, Listeria monocytogenes H7962 serotype 4, Escherichia coli O157:H4 FRIK 125, and E. coli ATCC 25922 were used in this study. The stock cultures of the pathogen species were frozen at −80 °C in broth media supplemented with glycerol (20% w/v). Stocks were transferred to tryptic soy agar (TSA) twice to activate the strains. S. Enteritidis KCCM 12021, E. coli O157:H4 FRIK 125, and E. coli ATCC 25922 were cultured in tryptic soy broth  at 37 °C for 18 h. L. monocytogenes H7962 serotype 4 was cultured in TSB supplemented with yeast extract (0.6% w/v) (TSB-YE) at 37 °C for 18 h.Four biofilm-forming pathogens, 5 CFU/mL. The cultures of E. coli O157:H4 FRIK 125 and E. coli ATCC 25922 were combined in a 1:1 ratio. The multi-species culture was adjusted such that the final concentration of each species was 1 × 105 CFU/mL.Four experimental groups were prepared after an 18 h incubation, depending on the bacterial species inoculated. Of these, three were mono-species cultures and the other was a multi-species culture. Each mono-species culture was adjusted to a final concentration of 1 × 10g for 15 min. The supernatant was filtered using a 0.45 μm pore size syringe filter , and the filtered fluid was used as the CJ.Raw chicken breast meat was purchased from retail outlets . Chicken juice (CJ) was prepared using the method described by Wang et al.  with som5 CFU/mL in the 96-well plates. The 96-well plates were incubated at 37 °C for 24 h, and the MIC was determined as the lowest concentration of ε-PL that inhibited bacterial growth.The MICs of ε-PL  were detected using a two-fold dilution method and TSB in 96-well polystyrene microtiter plates  . ε-PL wa5 CFU/mL each, using the same protocol as described in Salmonella, Oxford agar  supplemented with Oxford supplement (MB cell) for Listeria, Eosin methylene blue agar  for E. coli, and TSA for total cell counts .Time-kill assays were conducted by culturing bacterial cultures in TSB, with the same volume of ε-PL at concentrations of 1/2 × MIC, 1 × MIC, and 2 × MIC at 37 °C, and a non-treated medium was used as a control . Bacteri5 CFU/mL, and 50 μL of each was mixed in a 96-well plate with volume equivalent to that of ε-PL, with concentrations ranging from 1/2 × MIC to 2 × MIC, using a non-treated TSB as a control. Bacterial cultures were divided into four groups, as described in The biofilm assay was conducted using the method described by Yu et al. , with so5 CFU/mL, and 100 μL of each was incubated in 96-well plates at 37 °C for 24 h. The bacterial suspension was placed in 100 μL of ε-PL at concentrations of 1/2 × MIC to 2 × MIC, followed by incubation at 37 °C for 24 h. The subsequent crystal violet assay was conducted as described above. The biofilm formation rate (%) was determined using the following formula: treatment and ODcontrol are defined as the absorbance at 570 nm in a dissolving solution.To investigate the degradation effect of ε-PL on mature biofilms, bacterial cultures were adjusted to 1.0 × 10g at 4 °C for 5 min, and the cell supernatant was discarded. The pellet was washed twice with phosphate-buffered saline  and dissolved in CJ and diluted to a concentration of 1.0 × 105 CFU/mL. ε-PL was diluted in the CJ ranging from 1/2 × MIC to 2 × MIC. The same method was conducted as described above, except that TSB was substituted for CJ.To investigate the biofilm inhibition and degradation of ε-PL on CJ, the strains were cultured in broth medium at 37 °C for 18 h. The bacterial culture was centrifuged at 12000× 5 CFU/mL and prepared in four groups, as described in The bacterial surface hydrophobicity of food poisoning pathogens was investigated in the presence of ε-PL, following Yang et al.  with somg at 4 °C for 5 min, and the precipitates were rinsed twice. Collected cells were re-dissolved in PBS to adjust the OD600 to 0.5 ± 0.05 . Two milliliters of each suspension was mixed with chloroform (0.5 mL) and vortexed for 2 min. After being static at room temperature for 15 min, the upper aqueous layer was collected and measured at 600 nm (ODtreatment). The hydrophobicity (%) was calculated using the following equation:Cultured cells were centrifuged at 12,000× 5 CFU/mL and ε-PL solution at 1/2 × MIC was blended in a ratio of 1:1 and incubated at 37 °C for 24 h. Non-treated cells were used as control. Five milliliters of the mixture was collected and statically incubated at 4 °C for 24 h. After incubation, the upper aqueous layer was measured at 600 nm (ODtreatment). The sample was then vortexed and measured at 600 nm . Auto-aggregation (%) was calculated using the following equation:Auto-aggregation was conducted using a modification of the method presented by Kim et al. . Cell cug at 25 °C for 10 min and the precipitates were dispersed in 1.0 mL of 1.5 M sodium chloride. The cell emulsion was centrifuged at 5000× g at 25 °C for 10 min, and 60 μL of the supernatant was mixed with 60 μL of 5% phenol and 4 mL of sulfuric acid by vortexing. Then, the mixtures were incubated at 30 °C for 10 min, and the absorbance was measured at 490 nm. The total EPS production rate (%) was calculated using the following equation:treatment and ODcontrol were defined as the absorbance (OD490) of the phenol-sulfuric acid solution, treated with ε-PL and the control, respectively.The total EPS production rate of food-poisoning pathogens was determined according to Song et al. , with soCLSM was used to investigate the anti-biofilm effects of ε-PL against biofilm-forming pathogens, as previously described, with some modifications . Before 5 CFU/mL for cell suspension. Strains were cultured in appropriate broth media at 37 °C for 18 h, and their density was diluted using TSB to adjust the concentration to 1 × 10To investigate the inhibition effects on biofilm formation, two milliliters of cell suspension was transferred to each well of a 6-well plate  containing glass coupons. Two milliliters of ε-PL solution was adjusted to concentrations of 1 × MIC using TSB and added to each well of the plates. The control experiment used the non-treated medium. The 6-well plates were incubated at 37 °C for 24 h to form bacterial biofilms on the glass coupons. Then, the glass coupons were rinsed twice with PBS to remove planktonic cells. To investigate the eradication effects on mature biofilms, four milliliters of cell suspension was added to each well of a 6-well plate with glass coupons. After that, the 6-well plates were incubated at 37 °C for 24 h. Cell cultures were substituted to four milliliters of ε-PL solution at 1 × MIC, and then re-incubated for 37 °C for 24. The subsequent process was the same as that described above.For CLSM observation, each specimen was dyed as follows: 1 μM/mL each of SYTO9  and propidium iodide  were used to dye the living cells on glass coupons at room temperature for 20 min without light. Following staining, the glass coupons were rinsed twice with PBS and dried at room temperature for 30 min. The specimens were then investigated using a Zeiss LSM 800 microscope .The results are presented as the mean ± standard error. All experiments were conducted in triplicates. SPSS version 18.0  was used for statistical analysis. One-way analysis of variance (ANOVA) was used to determine the significance of the differences.S. Enteritidis KCCM 12021, L. monocytogenes H7962 serotype 4, E. coli O157:H4 FRIK 125, and E. coli ATCC 25922 were selected for this study. These species are representative of food-poisoning pathogens detected in poultry meat. In addition, they can form bacterial biofilms, which are a source of antimicrobial resistance [S. Enteritidis KCCM 12021, L. monocytogenes H7962 serotype 4, and E. coli were independently cultured, the MICs of ε-PL were 1.0, 0.031, and 1.0 mg/mL, respectively. The MIC of ε-PL in the co-cultured strain was 1.0 mg/mL. ε-PL has been shown to have antimicrobial effects against Gram-positive and Gram-negative bacteria, yeast, and fungi [S. Enteritidis and S. typhimurium, respectively [L. monocytogenes and E. coli showed MIC values at 125 and 19.53 μg/mL, respectively [The antimicrobial effect of ε-PL was estimated by investigating the MIC against the chosen foodborne pathogens . S. Entesistance ,28,29,30nd fungi ,33. Someectively . In addiS. Enteritidis KCCM 12021, L. monocytogenes H7962 serotype 4, and the E. coli group, cultured at 10 h incubation, reached 8.49 ± 0.09 log CFU/mL, 8.85 ± 0.01 log CFU/mL, and 8.94 ± 0.08 log CFU/mL, respectively. ε-PL at 1 × MIC showed bactericidal activity against all bacteria within 4 h, while at 1/2 × MIC showed bacteriostatic activity against all bacteria within 4 h except to L. monocytogenes. The E. coli group exhibited a gradual decline on the cell viability depending on incubation time. S. Enteritidis KCCM 12021 was diminished after 6 h of incubation and was extinguished at 10 h  after 4 h of treatment . S. Enteritidis KCCM 12021, L. monocytogenes H7962 serotype 4, E. coli, and multi-species strains at 1/2 × MIC were 28.12%, 35.87%, 60.38%, and 35.28%, respectively. Likewise, in the CJ medium, biofilm formation rates for S. Enteritidis KCCM 12021, L. monocytogenes H7962 serotype 4, the E. coli group, and multi-species strains at 1/2 × MIC were 10.36%, 9.79%, 24.73%, and 21.37%, respectively . However, the L. monocytogenes H7962 serotype 4 did not show a significant effect. Biofilm formation levels in the CJ medium were 50.41%, 9.1%, 26.73%, and 24.92% for S. Enteritidis KCCM 12021, L. monocytogenes H7962 serotype 4, the E. coli group, and multi-species strains at 1/2 × MIC (p < 0.05).Biofilm degradation affects the removal activity of mature biofilms of foodborne pathogens . The expectively B. ε-PL h/2 × MIC D. ε-PL sE. coli group. In addition, the biofilm formation rate of ε-PL in the CJ medium was generally lower than that in the TSB, for both the inhibitory and degradative effects of the biofilm. As biofilm formation and the bacterial cells remaining were both influenced by carbohydrates [Interestingly, ε-PL had inhibitory and degradative effects against bacterial biofilms at concentrations below the MIC, suggesting that ε-PL is an economical antibiofilm agent. Compared to the inhibitory effects of initial biofilm formation A,C with hydrates , there wSalmonella sp., which is associated with the potential molecular mechanisms [Salmonella sp. Other studies have reported the reduction in biofilms formed by Salmonella sp., P. aeruginosa, S. aureus, and L. monocytogenes when treated with ε-PL [Some researchers have demonstrated the anti-biofilm effects of ε-PL. ε-PL is a mediator of the inhibition of biofilm formation generated by chanisms . ε-PL doith ε-PL .L. monocytogenes H7962 serotype 4 treated with 1/2 × MIC of ε-PL showed significantly lower levels of hydrophobicity, auto-aggregation, and EPS production than the control group (without ε-PL), with decreased levels of 6.4%, 21.65%, and 25%, respectively. In addition, the EPS production (%) of multi-species culture at 1/2 × MIC of ε-PL was reduced by 43.71% as compared to that of the control group (p < 0.05). These results suggest that ε-PL inhibits the formation of biofilms of L. monocytogenes at the initial stage by weakening bacterial surface properties, and this characteristic is identically manifested in a co-culture of L. monocytogenes H7962 serotype 4 with Gram-negative strains in a limited area. However, S. Enteritidis, and E. coli strains did not show significant effects on bacterial surface properties or EPS production (data not shown).Hydrophobicity and auto-aggregation are the principal properties that form biofilms during the initial stage . Cell suS. Enteritidis KCCM 12021, L. monocytogenes H7962 serotype 4, and the E. coli group. The biofilm of the control showed abundant live colonies (green fluorescence), whereas biofilms treated with ε-PL showed few cells or increasing dead cells (red fluorescence). Some photographs of samples treated with 1 × MIC of ε-PL showed a rare-shaped structure due to the adsorption of ε-PL (Salmonella, Listeria, and E. coli, as also evidenced by the crystal violet assay results of the ε-PL antibiofilm activity (The inhibitory and degradation effects of ε-PL on the selective pathogen biofilms were optically analyzed using CLSM . Syto9 a of ε-PL e,g, wher of ε-PL c, which activity .S. Enteritidis, L. monocytogenes, and E. coli. ε-PL decreased cell viability depending on the time and concentration and induced the collapse of the cell membrane of pathogens, which proved the antibacterial effect of ε-PL. The antibiofilm effect of ε-PL was also evidenced by the reduction in biofilm formation and the destruction of the morphological form, as observed using CLSM. Antibiofilm effects were also observed in CJ. This study will help promote the safety of chicken meat processing, as it imitates the composition and microbiome of chicken meat to obtain more predictable results in contaminated environments. Therefore, ε-PL has the potential to be used as an antibiotic and antibiofilm material in the food industry, while accounting for the sanitary concerns of the consumers.This study demonstrated that ε-PL has antibacterial and antibiofilm effects against food poisoning pathogens detected in chicken, such as