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19 pages, 3108 KiB  
Article
Phytochemical Characterization of Bilberries and Their Potential as a Functional Ingredient to Mitigate Ochratoxin A Toxicity in Cereal-Based Products
by Denisia Pașca, Massimo Frangiamone, Luciano Mangiapelo, Pilar Vila-Donat, Oana Mîrza, Ana-Maria Vlase, Doina Miere, Lorena Filip, Jordi Mañes, Felicia Loghin and Lara Manyes
Nutrients 2024, 16(18), 3137; https://doi.org/10.3390/nu16183137 (registering DOI) - 17 Sep 2024
Abstract
Mycotoxin contamination of cereals and cereal-based products is a serious problem for food safety. Antioxidant-rich ingredients such as bilberries (Vaccinium myrtillus L., VM) may mitigate their harmful effects. Firstly, total phenolic content, antioxidant activity, and analytical phytochemical composition (hydroxycinnamic and hydroxybenzoic acids, [...] Read more.
Mycotoxin contamination of cereals and cereal-based products is a serious problem for food safety. Antioxidant-rich ingredients such as bilberries (Vaccinium myrtillus L., VM) may mitigate their harmful effects. Firstly, total phenolic content, antioxidant activity, and analytical phytochemical composition (hydroxycinnamic and hydroxybenzoic acids, flavanols, flavonols, and anthocyanins) were assessed in lyophilized wild bilberries from Romania. Secondly, this study evaluated bilberries’ effects on reducing ochratoxin A (OTA) bioaccessibility and cytotoxicity. An in vitro digestion model was developed and applied to four different types of bread: Control, VM (2%), OTA (15.89 ± 0.13 mg/kg), and OTA (16.79 ± 0.55 mg/kg)-VM (2%). The results indicated that VM decreased OTA bioaccessibility by 15% at the intestinal level. OTA-VM digests showed improved Caco-2 cell viability in comparison to OTA digests across different exposure times. Regarding the alterations in Jurkat cell line cell cycle phases and apoptosis/necrosis, significant increases in cell death were observed using OTA digests (11%), while VM addition demonstrated a protective effect (1%). Reactive oxygen species (ROS) analysis confirmed these findings, with OTA-VM digests showing significantly lower ROS levels compared to OTA digests, resulting in a 3.7-fold decrease. Thus, bilberries exhibit high potential as a functional ingredient, demonstrating protection in OTA mitigation effects. Full article
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Figure 1
<p>Chemical structure of OTA.</p>
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<p>Gastric and intestinal OTA bioaccessibility (%) calculated after the in vitro simulated digestion (n = 3). Significant differences between OTA and OTA-VM are indicated as <span class="html-italic">p</span> &lt; 0.05 (*), <span class="html-italic">p</span> &lt; 0.001 (***). Bread with Ochratoxin A (OTA); bread with Ochratoxin A + <span class="html-italic">Vaccinium myrtillus</span> L. 2% (OTA-VM).</p>
Full article ">Figure 3
<p>Cell viability in differentiated Caco-2 cells after exposure to various dilutions of intestinal digests (3.2 μM OTA for no dilution) over five different time points. The data are presented as mean ± SD (n = 4). Significant differences between OTA and OTA-VM intestinal digests at the same dilution and exposure time are denoted as <span class="html-italic">p</span> &lt; 0.05 (*); <span class="html-italic">p</span> &lt; 0.01 (**); <span class="html-italic">p</span> &lt; 0.001 (***). OTA: bread with wheat flour and barley contaminated with Ochratoxin A; OTA-VM: bread with wheat flour and barley flour contaminated with Ochratoxin A and <span class="html-italic">Vaccinium myrtillus</span> L. 2%.</p>
Full article ">Figure 4
<p>Effect of 1/10 intestinal bread digests (0.32 μM OTA) exposure for 7 days on Jurkat cells cycle phases (Sub G<sub>0</sub>/G<sub>1</sub>; G<sub>0</sub>/G<sub>1</sub>; S; G<sub>2</sub>/M). Data are presented as mean ± SD (n = 4) and significant differences between intestinal digests (VM, OTA, and OTA-VM) and control or OTA and OTA-VM are indicated as <span class="html-italic">p</span> &lt; 0.05 (*); <span class="html-italic">p</span> &lt; 0.01 (**); <span class="html-italic">p</span> &lt; 0.001 (***). The acronyms for cell cycle phases are G for growth, S for DNA synthesis, and M for mitosis. C: wheat flour bread; VM: bread with wheat flour and <span class="html-italic">Vaccinium myrtillus</span> L. 2%; OTA: bread with wheat flour and barley flour contaminated with Ochratoxin A; OTA-VM: bread with wheat flour and barley flour contaminated with Ochratoxin A and <span class="html-italic">Vaccinium myrtillus</span> L. 2%.</p>
Full article ">Figure 5
<p>Effect of intestinal digests (0.32 μM OTA) exposure on Jurkat cells after 7 days on the apoptosis and necrosis pathway. Significant differences between intestinal digests (VM, OTA, and OTA-VM) and control or OTA and OTA-VM are indicated as <span class="html-italic">p</span> &lt; 0.05 (*); <span class="html-italic">p</span> &lt; 0.001 (***) and data graph bars represent the mean ± SD (n = 4). C: wheat flour bread; VM: bread with wheat flour and <span class="html-italic">Vaccinium myrtillus</span> L. 2%; OTA: bread with wheat flour and barley flour contaminated with Ochratoxin A; OTA-VM: bread with wheat flour and barley flour contaminated with Ochratoxin A and <span class="html-italic">Vaccinium myrtillus</span> L. 2%.</p>
Full article ">Figure 6
<p>Reactive oxygen species (ROS) measured by flow cytometry using Jurkat cells after 7 days of exposure. (<b>A</b>) Effect of intestinal digests (0.32 μM OTA) on ROS generation. Mean fluorescence intensity is expressed as relative units with ± SD (n = 4). (<b>B</b>) MitoSOX-based flow cytometry detection of mitochondrial ROS in Jurkat cells following exposure to intestinal digests (0.32 μM OTA). Data in the histogram are presented as mean ± SD (n = 4). Significant differences between intestinal digests (VM, OTA, and OTA-VM) and the control, or between OTA and OTA-VM, are indicated as <span class="html-italic">p</span> &lt; 0.001 (***). C: wheat flour bread; VM: bread with wheat flour and <span class="html-italic">Vaccinium myrtillus</span> L. 2%; OTA: bread with wheat flour contaminated with Ochratoxin A; OTA-VM: bread with wheat flour contaminated with Ochratoxin A and <span class="html-italic">Vaccinium myrtillus</span> L. 2%; TBHP: tert-Butyl hydroperoxide.</p>
Full article ">Figure 7
<p>Effect of intestinal digest (0.32 μM OTA) on mitochondrial mass using Jurkat cells after 7 days of exposure. Median fluorescence intensity (MFI) (n = 4) of MitoTracker dye measured after incubation of Jurkat cells with intestinal digests. Significant differences between intestinal digests (VM, OTA, and OTA-VM) and control or OTA and OTA-VM are indicated as <span class="html-italic">p</span> &lt; 0.001 (***). C: wheat flour bread; VM: bread with wheat flour and <span class="html-italic">Vaccinium myrtillus</span> L. 2%; OTA: bread with wheat flour contaminated with Ochratoxin A; OTA-VM: bread with wheat flour contaminated with Ochratoxin A and <span class="html-italic">Vaccinium myrtillus</span> L. 2%.</p>
Full article ">
18 pages, 1030 KiB  
Article
Effect of Low-Proportion Replacement of Dietary Fishmeal with Neanthes japonica Meal on Growth Performance, Body Composition, Muscle Texture, Serum Biochemistry, Digestive Enzymes and Gene Expression in Juvenile Tiger Puffer Takifugu rubripes
by Qingyan Gao, Yuhan Fan, Renxiao Zhang, Jinghui Fang, Qiang Ma, Yuliang Wei, Mengqing Liang, Feng Liu and Houguo Xu
Fishes 2024, 9(9), 362; https://doi.org/10.3390/fishes9090362 (registering DOI) - 17 Sep 2024
Abstract
The polychaeta Neanthes japonica is a common by-product in mariculture ponds. It is rich in essential nutrients, but has not been well-explored. Therefore, this 56-day experiment investigated the effects of substituting N. japonica meal for dietary fishmeal on juvenile tiger puffer (15.49 ± [...] Read more.
The polychaeta Neanthes japonica is a common by-product in mariculture ponds. It is rich in essential nutrients, but has not been well-explored. Therefore, this 56-day experiment investigated the effects of substituting N. japonica meal for dietary fishmeal on juvenile tiger puffer (15.49 ± 0.02 g, n = 450 fish). The control diet (CON) contained 40% fishmeal. Freeze-dried N. japonica meal (FNM) was supplemented into CON at the levels of 3% (FNM3), 6% (FNM6), and 9% (FNM9), replacing fishmeal. The fifth diet contained 6% oven-dried N. japonica meal (ONM6), replacing fishmeal. The results indicated that no significant difference was observed in growth, feed efficiency, and somatic index among all the treatment groups. The feed intake of the FNM6 group was significantly higher compared to CON. No significant difference was detected in fish proximate composition, as well as the fatty acid composition, amino acid composition, and muscle texture. The supplementation of N. japonica meal decreased the activities of intestinal lipase and α-amylase. The addition of freeze-dried N. japonica meal significantly up-regulated the expression of the intestinal amino-glycine transporter pat1. It was concluded that adding 9% N. japonica meal to the feed had no significant effect on the growth performance and body composition of juvenile tiger puffer. Full article
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Figure 1
<p>Effects of dietary <span class="html-italic">N. japonica</span> meal on the total protein (TP, (<b>A</b>)), triglycerides (TG, (<b>B</b>)), total cholesterol (TC, (<b>C</b>)), malondialdehyde (MDA, (<b>D</b>)), lysozyme (LZM, (<b>E</b>)), and albumin (ALB, (<b>F</b>)) in serum of tiger puffer. Data bars not sharing the same letter are significantly different (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 2
<p>Effects of dietary <span class="html-italic">N. japonica</span> meal on the trypsin (<b>A</b>), chymotrypsin (<b>B</b>), lipase (<b>C</b>), total amylase (<b>D</b>), β-amylase (<b>E</b>), and α-amylase (<b>F</b>) activity in the intestine of tiger puffer. Data bars not sharing the same letter are significantly different (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 3
<p>Effects of dietary <span class="html-italic">N. japonica</span> meal on the gene expression of peptide and amino acid transporters in the intestine of tiger puffer (<b>A</b>). Effects of dietary <span class="html-italic">N. japonica</span> meal on the gene expression of <span class="html-italic">leptin</span> and JAK-STAT signaling pathway in the liver and intestine of tiger puffer (<b>B</b>). Data bars not sharing the same letter are significantly different (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">
15 pages, 1585 KiB  
Article
Development of Potentially Probiotic Mead from Co-Fermentation by Saccharomyces cerevisiae var. boulardii and Kombucha Microorganisms
by Handray Fernandes de Souza, Eduardo Novais Souza Freire, Giovana Felício Monteiro, Lorena Teixeira Bogáz, Ricardo Donizete Teixeira, Fabiano Vaquero Silva Junior, Felipe Donizete Teixeira, João Vitor dos Santos, Marina Vieira de Carvalho, Ramon da Silva Rocha, Adriano Gomes da Cruz, Juliana Maria Leite Nobrega de Moura Bell, Igor Viana Brandi and Eliana Setsuko Kamimura
Fermentation 2024, 10(9), 482; https://doi.org/10.3390/fermentation10090482 (registering DOI) - 17 Sep 2024
Viewed by 137
Abstract
Mead is a fermented alcoholic beverage produced from a diluted solution of honey and yeast activity. The objectives of this study were to produce a potentially probiotic mead through mixed fermentation by Saccharomyces cerevisiae var. boulardii and kombucha microorganisms and to evaluate fermentation [...] Read more.
Mead is a fermented alcoholic beverage produced from a diluted solution of honey and yeast activity. The objectives of this study were to produce a potentially probiotic mead through mixed fermentation by Saccharomyces cerevisiae var. boulardii and kombucha microorganisms and to evaluate fermentation kinetics, microbial cell survival and their in vitro resistance to simulated gastrointestinal transit, color parameters and the phenolic and antioxidant potential of the product. The main results of this study show that in order to develop a potentially probiotic mead utilizing the mixed fermentation of S. boulardii and kombucha microorganisms, the best condition was a concentration of 25 mL/L (v/v) of kombucha and 0.75 g/L (w/v) of S. boulardii with fermentation for 9 days at a temperature of 25 °C. In addition, at the end of fermentation, mead with kombucha and S. boulardii presented physicochemical characteristics with a pH of 3.48, 0.67% total acidity, 18.76 °Brix soluble solids and 4.77% alcohol content. The S. boulardii and lactic acid bacteria (LAB) present in the mead survived conditions reproducing those of the gastrointestinal tract, with counts of more than 6 Log10 CFU/mL for both microorganisms after the intestinal phase. In the color analysis, the mead with kombucha and S. boulardii had a yellow color with the b* parameter corresponding to 35.93, luminosity (L*) equal to 76.09 and 1.82 for a*. In addition, the mead we produced contains quantities of phenolics and antioxidants. In conclusion, kombucha and S. boulardii are presented as alternative microbial sources for obtaining potentially probiotic mead. Full article
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Figure 1

Figure 1
<p>Kombucha obtained from green tea (<span class="html-italic">Camellia sinensis</span>), after 20 days of fermentation, at a temperature of 25 °C. scoby = symbiotic culture of bacteria and yeast.</p>
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<p>Mead fermentation kinetics over 9 days at a temperature of 25 °C. (<b>A</b>) pH, (<b>B</b>) Total acidity (% lactic acid), (<b>C</b>) Soluble solids (°Brix) and (<b>D</b>) Alcohol content (%). T1 = mead with kombucha and <span class="html-italic">S. boulardii</span>; T2 = mead with kombucha and commercial <span class="html-italic">S. cerevisiae</span>. Different letters indicate that there are significant differences between the treatments when the Tukey test is applied (<span class="html-italic">p</span> &gt; 0.05).</p>
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<p>Viable cell count in mead and simulated in vitro digestibility at 37 °C. (<b>A</b>) Yeast count (Log<sub>10</sub> CFU/mL), (<b>B</b>) Yeast survival rate (%), (<b>C</b>) Lactic acid bacteria count (Log<sub>10</sub> CFU/mL) and (<b>D</b>) Lactic acid bacteria survival rate (%). T1 = mead with kombucha and <span class="html-italic">S. boulardii</span>; T2 = mead with kombucha and commercial <span class="html-italic">S. cerevisiae</span>. Different lowercase letters indicate that there is a significant difference between the treatments. Different capital letters above the bars indicate that there are significant differences between the different phases, for each treatment. The Tukey test (<span class="html-italic">p</span> &gt; 0.05) was applied to the difference in means.</p>
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<p>Visual appearance and color of the meads produced. T1 = mead with kombucha and <span class="html-italic">S. boulardii</span>; T2 = mead with kombucha and commercial <span class="html-italic">S. cerevisiae</span>. R1, R2 and R3 = repetitions 1, 2 and 3, respectively.</p>
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20 pages, 3229 KiB  
Article
Pterostilbene, a Dimethyl Derivative of Resveratrol, Exerts Cytotoxic Effects on Melanin-Producing Cells through Metabolic Activation by Tyrosinase
by Hitomi Tanaka, Tomoko Nishimaki-Mogami, Norimasa Tamehiro, Norihito Shibata, Hiroki Mandai, Shosuke Ito and Kazumasa Wakamatsu
Int. J. Mol. Sci. 2024, 25(18), 9990; https://doi.org/10.3390/ijms25189990 (registering DOI) - 17 Sep 2024
Viewed by 168
Abstract
Pterostilbene (PTS), which is abundant in blueberries, is a dimethyl derivative of the natural polyphenol resveratrol (RES). Several plant species, including peanuts and grapes, also produce PTS. Although RES has a wide range of health benefits, including anti-cancer properties, PTS has a robust [...] Read more.
Pterostilbene (PTS), which is abundant in blueberries, is a dimethyl derivative of the natural polyphenol resveratrol (RES). Several plant species, including peanuts and grapes, also produce PTS. Although RES has a wide range of health benefits, including anti-cancer properties, PTS has a robust pharmacological profile that includes a better intestinal absorption and an increased hepatic stability compared to RES. Indeed, PTS has a higher bioavailability and a lower toxicity compared to other stilbenes, making it an attractive drug candidate for the treatment of various diseases, including diabetes, cancer, cardiovascular disease, neurodegenerative disorders, and aging. We previously reported that RES serves as a substrate for tyrosinase, producing an o-quinone metabolite that is highly cytotoxic to melanocytes. The present study investigated whether PTS may also be metabolized by tyrosinase, similarly to RES. PTS was oxidized as a substrate by tyrosinase to form an o-quinone, which reacted with thiols, such as N-acetyl-L-cysteine, to form di- and tri-adducts. We also confirmed that PTS was taken up and metabolized by human tyrosinase-expressing 293T cells in amounts several times greater than RES. In addition, PTS showed a tyrosinase-dependent cytotoxicity against B16BL6 melanoma cells that was stronger than RES and also inhibited the formation of melanin in B16BL6 melanoma cells and in the culture medium. These results suggest that the two methyl groups of PTS, which are lipophilic, increase its membrane permeability, making it easier to bind to intracellular proteins, and may therefore be more cytotoxic to melanin-producing cells. Full article
(This article belongs to the Collection Feature Papers in Bioactives and Nutraceuticals)
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Figure 1
<p>Structures of resveratrol (RES, <b>1</b>) and pterostilbene (PTS, <b>2</b>).</p>
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<p>Scheme showing the tyrosinase-catalyzed oxidation of pterostilbene (PTS, <b>2</b>) in the absence or presence of a thiol. The oxidation of PTS (<b>2</b>) gives PTS-quinone (<b>3</b>) as an immediate product, which rapidly decays. PTS-quinone (<b>3</b>) is reduced by ascorbic acid (AA) to form PTS-catechol (3′-hydroxyPTS, (<b>4</b>)). The tyrosinase-catalyzed oxidation of PTS (<b>2</b>) in the presence of the thiol <span class="html-italic">N</span>-acetyl-L-cysteine (NAC) affords the di-adduct DiNAC-PTS-catechol (<b>5</b>) and the tri-adduct TriNAC-PTS-catechol (<b>6</b>). These thiol adducts were isolated and identified as the NAC adducts; “+” means that DiNAC-PTS-catechol (<b>5</b>) and TriNAC-PTS-catechol (<b>6</b>) are produced together.</p>
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<p>Time course of the tyrosinase-catalyzed oxidation of PTS (<b>2</b>) and PTS-catechol (<b>4</b>) and HPLC analyses of reaction products. (<b>a</b>) UV/visible spectral changes of PTS (<b>2</b>) at pH 6.8. (<b>b</b>) HPLC analysis following the tyrosinase-catalyzed oxidation of PTS (<b>2</b>) at pH 6.8, the reaction being stopped by the addition of NaBH<sub>4</sub>, followed by HClO<sub>4</sub>. The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and is representative.</p>
Full article ">Figure 4
<p>Time course of the tyrosinase-catalyzed oxidation of PTS (<b>2</b>) in presence of ascorbic acid (AA). (<b>a</b>) UV/visible spectral changes of PTS (<b>2</b>) at pH 6.8 in presence of 10 mol eq. AA. (<b>b</b>) HPLC analysis following the tyrosinase-catalyzed oxidation of PTS (<b>2</b>) at pH 6.8 in presence of 10 mol eq. AA, the reaction being stopped by the addition of NaBH<sub>4</sub>, followed by HClO<sub>4</sub>. The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and is representative.</p>
Full article ">Figure 5
<p>Time course of the tyrosinase-catalyzed oxidation of PTS (<b>2</b>) in presence of NAC. (<b>a</b>) UV/visible spectral changes of PTS (<b>2</b>) in presence of 2 mol eq. NAC at pH 6.8. (<b>b</b>) UV/visible spectral changes of PTS (<b>2</b>) in presence of 3 mol eq. NAC at pH 6.8. The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and is representative.</p>
Full article ">Figure 6
<p>Time course of the tyrosinase-catalyzed oxidation of PTS (<b>2</b>) in presence of NAC. (<b>a</b>) HPLC chromatogram of the tyrosinase-catalyzed oxidation of PTS (<b>2</b>) for 5 min in presence of 3 mol eq. NAC at pH 6.8, the reaction being stopped by the addition of NaBH<sub>4</sub>, followed by HClO<sub>4</sub>. Peak: #1; tri-adduct (<b>6</b>), #2; di-adduct (<b>5</b>), #3; PTS-catechol (<b>4</b>), #4; PTS (<b>2</b>). HPLC analyses were performed at 45 °C at a flow rate of 0.7 mL/min. (<b>b</b>) HPLC analysis following the tyrosinase-catalyzed oxidation of PTS (<b>2</b>) in presence of 3 mol eq. NAC at pH 6.8, the reaction being stopped by the addition of NaBH<sub>4</sub>, followed by HClO<sub>4</sub>. The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and is representative.</p>
Full article ">Figure 7
<p>Metabolism of RES (<b>1</b>) and PTS (<b>2</b>) in hTYR-293T cells yielding their adducts with CySH and GSH. (<b>a</b>) RES (<b>1</b>) and its metabolites in cells. (<b>b</b>) PTS (<b>2</b>) and its metabolites in cells. (<b>c</b>) RES (<b>1</b>) and its metabolites in the medium. (<b>d</b>) PTS (<b>2</b>) and its metabolites in the medium. Data represent means ± SD (<span class="html-italic">n</span> = 3 wells). Statistically significant differences: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001 between each treatment concentration (in µM) at RES (<b>1</b>) and PTS (<b>2</b>). The statistically significance of the differences was determined by Student’s <span class="html-italic">t</span>-test (two-tailed).</p>
Full article ">Figure 8
<p>(<b>a</b>) Level of <span class="html-italic">Tyr</span> mRNA in siRNA-transfected cells. (<b>b</b>) 4SCAP, (<b>c</b>) RES (<b>1</b>), and (<b>d</b>) PTS (<b>2</b>) induce tyrosinase-dependent reductions in the viability of B16BL6 melanoma cells. B16BL6 melanoma cells were transfected with a negative control siRNA or with a siRNA directed against <span class="html-italic">Tyr</span> for 24 h. The cells were then treated with the indicated concentrations of compounds for 24 h, and their viability and mRNA levels (in vehicle-treated cells) were measured. Data represent means ± SD (<span class="html-italic">n</span> = 3 wells). The experiments were repeated once, and good reproducibility was obtained. The figure was from a single experiment and representative. * <span class="html-italic">p</span> &lt; 0.05 between siRNA control and siRNA <span class="html-italic">Tyr</span> at each treatment concentration (in µM). #1: <span class="html-italic">p</span> = 0.017 between RES (<b>1</b>) and PTS (<b>2</b>) in siRNA Ctrl. #2: <span class="html-italic">p</span> = 0.020 between RES (<b>1</b>) and PTS (<b>2</b>) in siRNA <span class="html-italic">Tyr</span> at 100 µM, respectively.</p>
Full article ">Figure 9
<p>(<b>a</b>,<b>b</b>): Eumelanin and pheomelanin contents in B16BL6 melanoma cells. Data represent means ± SD (<span class="html-italic">n</span> = 3 wells). <span class="html-italic">p</span> value between control (0 μM) and each treatment concentration (µM) at eumelanin and pheomelanin in B16BL6 melanoma cells. Statistically significant differences: * <span class="html-italic">p</span> &lt; 0.01, ** <span class="html-italic">p</span> &lt; 0.001. (<b>c</b>,<b>d</b>): Eumelanin and pheomelanin contents in the medium. <span class="html-italic">p</span> value between control (0 μM) and each treatment concentration (µM) at eumelanin and pheomelanin in the medium. * <span class="html-italic">p</span> &lt; 0.01, ** <span class="html-italic">p</span> &lt; 0.001. #1: <span class="html-italic">p</span> = 0.026 between RES (<b>1</b>) and PTS (<b>2</b>) in eumelanin values at 3 µM. #2: <span class="html-italic">p</span> = 0.002 between RES (<b>1</b>) and PTS (<b>2</b>) in pheomelanin values at 3 µM.</p>
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10 pages, 7573 KiB  
Communication
Rapid Purification and Quantification of Intestinal and Fecal Short-Chain Fatty Acids by Solid-Phase Extraction Using Bond Elut Plexa
by Xing Zheng, Tao Chen, Wanli Li, Kai Wang, Xiaofeng Xue, Nenad Naumovski and Wenjun Peng
Separations 2024, 11(9), 270; https://doi.org/10.3390/separations11090270 - 16 Sep 2024
Viewed by 301
Abstract
Short-chain fatty acids (SCFAs) in feces are inextricably linked to intestinal homeostasis and can be used as potential markers for metabolic diseases. In this study, an efficient and simple method was developed for the purification of SCFAs without the need for derivatization of [...] Read more.
Short-chain fatty acids (SCFAs) in feces are inextricably linked to intestinal homeostasis and can be used as potential markers for metabolic diseases. In this study, an efficient and simple method was developed for the purification of SCFAs without the need for derivatization of the samples. The SCFAs (acetic, propionic, isobutyric, butyric, isovaleric, valeric, and hexanoic acid) were extracted from a small amount (50 mg) of fecal and intestinal samples using acetone combined with solid phase extraction column (Bond Elut Plexa) enrichment. Quantitative analysis was performed using gas chromatography with a flame ionization detector. The developed method has shown very good limits of detection (LOD, 0.11–0.36 μM) and limits of quantification (LOQ, 0.38–1.21 μM) with excellent linearity (R2 ≥ 0.9998), good recovery (98.34–137.83%), and high reproducibility (RSD ≤ 1.30). The applicability of this method was also demonstrated by testing the fecal and cecum contents of different species from mammals (mice, pigs) to insects (honeybees). The technique is highly suitable for analyzing complex, small amounts of intestinal and fecal SCFAs. Full article
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Graphical abstract

Graphical abstract
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<p>Flow chart and theory of extracting SCFA from fecal samples.</p>
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<p>Chromatogram of mixed standards at L3 concentration (representing the average physiological concentration of SCFAs in the fecal and intestinal samples). Acetic acid, 107.41 µM; Propionic acid, 80.32 µM; Isobutyric acid, 63.11 µM; Butyric acid 64.92 µM; Isovaleric acid, 53.56 µM; Valeric acid, 54.34 µM; Hexanoic acid, 45.89 µM.</p>
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<p>(<b>A</b>) Gas chromatograms of standards and samples. (a) SCFA standards of L3 concentration; (b) SCFA extract from mouse cecum contents; (c) SCFA extract from mouse feces; (d) SCFA extract from pig cecum contents; (e) SCFA extract from pig feces; (f) SCFA extract from honeybees’ (Apis mellifera) intestinal contents. Numbers 1−7 represent acetic, propionic, isobutyric, butyric, isovaleric, valeric and hexanoic acid, respectively. (<b>B</b>) The PCA plots of the SCFA data in fecal and intestinal content samples from different sources. MCC, mouse cecum contents; MF, mouse feces; PCC, pig cecum contents; PF, pig feces; HIC, honeybee intestinal contents. (<b>C</b>) To compare the levels of acetic, propionic, isobutyric, butyric, isovaleric, valeric, and hexanoic acids in different samples. Statistical data are expressed as mean ± standard deviation. The results corresponding to bars with different letters were statistically significant (<span class="html-italic">p</span> &lt; 0.05). On the contrary, the results corresponding to bars with the same letter were not statistically significant.</p>
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15 pages, 2922 KiB  
Article
Specific Synbiotic Sugars Stimulate Streptococcus salivarius BLIS K12 and BLIS M18 Lantibiotic Production to Expand Bacterial Inhibition Range and Potency
by Liam K. Harold, Nicola C. Jones, Sarah L. Barber, Abigail L. Voss, Rohit Jain, John R. Tagg and John D. F. Hale
Appl. Microbiol. 2024, 4(3), 1320-1334; https://doi.org/10.3390/applmicrobiol4030091 (registering DOI) - 16 Sep 2024
Viewed by 176
Abstract
Synbiotics are mixtures of prebiotics and probiotics that enhance the activity of probiotic bacteria when co-administered to provide greater benefits to the host. Traditionally, the synbiotics that have been discovered enhance gut probiotic strains and are nutritionally complex molecules that survive digestive breakdown [...] Read more.
Synbiotics are mixtures of prebiotics and probiotics that enhance the activity of probiotic bacteria when co-administered to provide greater benefits to the host. Traditionally, the synbiotics that have been discovered enhance gut probiotic strains and are nutritionally complex molecules that survive digestive breakdown until they reach the later stages of the intestinal tract. Here, we screened and identified sugars or sugar substitutes as synbiotics for the oral probiotic strains Streptococcus salivarius BLIS K12 and BLIS M18. Using a modified deferred antagonism assay, we found that 0.5% (w/v) galactose and 2.5% (w/v) raffinose were the best candidates for use as synbiotics with BLIS K12 and M18, as they trigger enhanced antimicrobial activity against a range of bacteria representing species from the mouth, gut, and skin. Using reverse transcriptase quantitative PCR, we found that this enhanced antimicrobial activity was caused by the upregulation of the lantibiotic genes salA, salB, and sal9 in either K12 or M18. This led to the conclusion that either 2.5% (w/v) raffinose or 0.5% (w/v) galactose, respectively, are suitable synbiotics for use in conjunction with BLIS K12 and M18 to enhance probiotic performance. Full article
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<p>(<b>A</b>) Sum of the mean zones of inhibition for different sugars (left Y-axes, Bars) and the number of species inhibited (right Y-axes, square points) for BLIS K12 and BLIS M18 at both 2.5% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) and 0.5% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) of different sugars (Data points are mean (n = 3, ±SD). For the sum of the average mean zones of inhibition, a two-way ANOVA with Dunnett’s multiple comparisons tests compared raffinose in the 2.5% samples and galactose in 0.5% to the other sugars was completed. * = <span class="html-italic">p</span> &lt; 0.05 for all sugars, # = <span class="html-italic">p</span> &lt; 0.05 for all sugars except lactulose and lactose and ^ = <span class="html-italic">p</span> &lt; 0.05 for all sugar except raffinose. (<b>B</b>) Mean growth of BLIS K12 and BLIS M18 in M17 media supplemented with different types and concentrations of sugars after 18 h. (n = 3, ±SD). (<b>C</b>) Dose response of raffinose and galactose at different concentrations measuring the mean increase in zones of inhibition and number of species of bacteria inhibited compared to a non-sugar control across <span class="html-italic">S. constellatus</span> T29, <span class="html-italic">S. salivarius</span> K34b, <span class="html-italic">S. pyogenes</span> 71-698, <span class="html-italic">S. mutans</span> OMZ175, <span class="html-italic">S. pneumoniae</span> D39, <span class="html-italic">S. saprophyticus</span> ATCC 13505, <span class="html-italic">S. aureus</span> A222, <span class="html-italic">S. sobrinus</span> OMZ176. Lines with dots represent the mean increase in the size of the inhibition zone with a line of non-linear fit (left Y axes) and bars represent the number of species inhibited (right Y axes).</p>
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<p>Size of the inhibition zone for BLIS K12 and BLIS M18 on CABCa agar plates, containing 2.5% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) raffinose or 0.5% (<span class="html-italic">w</span>/<span class="html-italic">v</span>) galactose or no sugar, across a range of bacterial species and strains associated with (<b>A</b>) pharyngitis, otitis media, pneumonia, and neonatal sepsis infections. (<b>B</b>) Teeth (<b>C</b>) periodontitis, halitosis, and gut (<b>D</b>) skin. Bars are means of biological replicates (n = 3, ±SD). (<b>A</b>–<b>D</b>) A two-way ANOVA with Dunnett’s multiple comparisons tests compared raffinose and galactose to the no sugar control. *, <span class="html-italic">p</span> &lt; 0.05, **, <span class="html-italic">p</span> &lt; 0.01, ***, <span class="html-italic">p</span> &lt; 0.001, ****, <span class="html-italic">p</span> &lt; 0.0001.</p>
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<p>Mean expression levels of (<b>A</b>) <span class="html-italic">salA</span> (<b>B</b>) <span class="html-italic">salB</span> in BLIS K12 and (<b>C</b>) <span class="html-italic">salA</span> (<b>D</b>) <span class="html-italic">sal9</span> in BLIS M18, when grown on CABCa agar medium supplemented with different sugars relative to a no sugar control. Bars represent mean values with individual values shown as symbols (n = 3, ±SD).</p>
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<p>Illustrates the potential mechanisms by which the synbiotic sugars, raffinose and galactose enhance the probiotic performance of BLIS K12 and BLIS M18. Created with Created in BioRender. Hale, J. (2024) <a href="http://BioRender.com/g80w234" target="_blank">BioRender.com/g80w234</a> (accessed on 13 September 2024).</p>
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9 pages, 232 KiB  
Communication
Regenerating Family Member 3 Alpha Is Predictive of Mortality Following Emergent Large Vessel Occlusion
by Madison Sands, Christopher J. McLouth, Jacqueline A. Frank, Benton Maglinger, Nathan Millson, Mais N. Al-Kawaz, Shivani Pahwa, David L. Dornbos, Douglas E. Lukins, Amanda L. Trout, Ann M. Stowe, Justin F. Fraser and Keith R. Pennypacker
Int. J. Mol. Sci. 2024, 25(18), 9968; https://doi.org/10.3390/ijms25189968 (registering DOI) - 16 Sep 2024
Viewed by 220
Abstract
Regenerating Family Member 3 Alpha (REG3A) is an antimicrobial protein secreted by the intestine and pancreas with additional immunomodulatory properties. Previously, we published that REG3A expression in ischemic stroke patient systemic blood, during mechanical thrombectomy (MT), is significantly associated with inflammatory cytokines and [...] Read more.
Regenerating Family Member 3 Alpha (REG3A) is an antimicrobial protein secreted by the intestine and pancreas with additional immunomodulatory properties. Previously, we published that REG3A expression in ischemic stroke patient systemic blood, during mechanical thrombectomy (MT), is significantly associated with inflammatory cytokines and patient function on admission. This paper, however, did not investigate post-acute death rates. Therefore, we investigated plasma REG3A protein expression, during MT, in patients (n = 141) that survived or died within the end of the follow-up after MT. Subjects who died had significantly higher systemic plasma REG3A levels at the time of MT compared to survivors (p = 0.001). Age, sex, time from last known normal, and admission NIHSS were included as predictors to control for confounding variables and were all examined to determine their association in patient mortality. Logistic regression was used to demonstrate that higher odds of death were associated with increased REG3A levels (p = 0.002). REG3A demonstrated acceptable discrimination (AUC (95% CI): 0.669 (0.566–0.772) in predicting mortality. The overall model with age, sex, time from last known normal, and admission NIHSS discriminated well between survivors and those who died (AUC (95% CI): 0.784 (0.703–0.864)). In conclusion, REG3A could be promising as a biomarker to prognosticate stroke outcomes and stratify high-risk groups following acute ischemic stroke. Full article
(This article belongs to the Special Issue Proteomics and Its Applications in Disease 3.0)
16 pages, 5561 KiB  
Article
Metagenomic and Metabolomic Analyses Reveal the Role of Gut Microbiome-Associated Metabolites in the Muscle Elasticity of the Large Yellow Croaker (Larimichthys crocea)
by Zhenheng Cheng, Hao Huang, Guangde Qiao, Yabing Wang, Xiaoshan Wang, Yanfeng Yue, Quanxin Gao and Shiming Peng
Animals 2024, 14(18), 2690; https://doi.org/10.3390/ani14182690 - 16 Sep 2024
Viewed by 275
Abstract
The large yellow croaker (LYC, Larimichthys crocea) is highly regarded for its delicious taste and unique flavor. The gut microbiota has the ability to affect the host muscle performance and elasticity by regulating nutrient metabolism. The purpose of this study is to [...] Read more.
The large yellow croaker (LYC, Larimichthys crocea) is highly regarded for its delicious taste and unique flavor. The gut microbiota has the ability to affect the host muscle performance and elasticity by regulating nutrient metabolism. The purpose of this study is to establish the relationship between muscle quality and intestinal flora in order to provide reference for the improvement of the muscle elasticity of LYC. In this study, the intestinal contents of high muscle elasticity males (IEHM), females (IEHF), and low muscle elasticity males (IELM) and females (IELF) were collected and subjected to metagenomic and metabolomic analyses. Metagenomic sequencing results showed that the intestinal flora structures of LYCs with different muscle elasticities were significantly different. The abundance of Streptophyta in the IELM (24.63%) and IELF (29.68%) groups was significantly higher than that in the IEHM and IEHF groups. The abundance of Vibrio scophthalmi (66.66%) in the IEHF group was the highest. Based on metabolomic analysis by liquid chromatograph-mass spectrometry, 107 differentially abundant metabolites were identified between the IEHM and IELM groups, and 100 differentially abundant metabolites were identified between the IEHF and IELF groups. Based on these metabolites, a large number of enriched metabolic pathways related to muscle elasticity were identified. Significant differences in the intestinal metabolism between groups with different muscle elasticities were identified. Moreover, the model of the relationship between the intestinal flora and metabolites was constructed, and the molecular mechanism of intestinal flora regulation of the nutrient metabolism was further revealed. The results help to understand the molecular mechanism of different muscle elasticities of LYC and provide an important reference for the study of the mechanism of the effects of LYC intestinal symbiotic bacteria on muscle development, and the development and application of probiotics in LYC. Full article
(This article belongs to the Section Animal Genetics and Genomics)
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<p>Muscle elastic characteristics of LYCs.</p>
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<p>Agarose gel electrophoresis for sex determination in LYCs.</p>
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<p>Structural analysis of the LYC intestinal flora. (<b>a</b>) Male LYC intestinal flora at the phylum level; (<b>b</b>) Female LYC intestinal flora at the phylum level; (<b>c</b>) Male LYC intestinal flora at the genus level; (<b>d</b>) Female LYC intestinal flora at the genus level; (<b>e</b>) Male LYC intestinal flora at the species level; (<b>f</b>) Female LYC intestinal flora at the species level; (<b>g</b>) Male LYC intestinal flora Alpha diversity analysis; (<b>h</b>) Female LYC intestinal flora Alpha diversity analysis. * <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>Analysis of the differences in the gut flora of LYC with different muscle elasticities: (<b>a</b>) Venn diagrams between the IEHM and IELM groups at the genus level; (<b>b</b>) Non-metric multidimensional scaling (NMDS) analysis of the intestinal flora in male LYCs; (<b>c</b>) Principal coordinate analysis (PCoA) of the intestinal flora in male LYCs; (<b>d</b>) Principal component analysis (PCA) of the intestinal flora in male LYCs; (<b>e</b>) Venn diagrams between the IEHF and IELF groups at the genus level.; (<b>f</b>) Non-metric multidimensional scaling (NMDS) analyses of the intestinal flora in female LYCs; (<b>g</b>) Principal coordinate analysis (PCoA) of the intestinal flora in female LYCs; (<b>h</b>) Principal component analysis (PCA) of the intestinal flora in female LYCs.</p>
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<p>Heat map of the relative abundance of the gut flora. (<b>a</b>) Heat map of the relative abundance of the gut flora in males at the phylum level; (<b>b</b>) Heat map of the relative abundance of the gut flora in females at the phylum level; (<b>c</b>) Heat map of the relative abundance of the gut flora in males at the genus level; (<b>d</b>) Heat map of the relative abundance of the gut flora in females at the genus level; (<b>e</b>) Heat map of the relative abundance of the gut flora in males at the species level; (<b>f</b>) Heat map of the relative abundance of the gut flora in females at the species level; LYC, large yellow croaker; LYC, large yellow croaker; IEHM, intestinal contents of high muscle elasticity males; IELM, intestinal contents of low muscle elasticity males; IEHF, intestinal contents of high muscle elasticity females; IELF, intestinal contents of low muscle elasticity females.</p>
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<p>Analysis of LYC intestinal metabolites: (<b>a</b>) Intestinal metabolite analysis in male LYCs (Pos, positive ion mode); (<b>b</b>) Intestinal metabolite analysis in male LYCs (Neg, negative ion mode); (<b>c</b>) Intestinal metabolite analysis in female LYCs (positive ion mode); (<b>d</b>) Intestinal metabolite analysis in female LYCs (negative ion mode); (<b>e</b>) Differential metabolite analysis of LYC intestinal contents (positive ion mode); (<b>f</b>) Differential metabolite analysis of LYC intestinal contents (negative ion mode).</p>
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<p>Analysis of LYC intestinal metabolites: (<b>a</b>) Random forest analysis of intestinal metabolites in male LYCs; (<b>b</b>) Random forest analysis of intestinal metabolites in female LYCs; (<b>c</b>) Differential metabolite enriched pathway analysis of intestinal contents in male LYCs; (<b>d</b>) Differential metabolite enriched pathway analysis of intestinal contents in female LYCs. LYC, large yellow croaker; IEHM, intestinal contents of high muscle elasticity males; IELM, intestinal contents of low muscle elasticity males; IEHF, intestinal contents of high muscle elasticity females; IELF, intestinal contents of low muscle elasticity females; PC2, principal component 2.</p>
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<p>Association analysis of the LYC gut flora with differentially abundant metabolites. (<b>a</b>) male LYCs; (<b>b</b>) female LYCs. LYC, large yellow croak. The symbol * is the degree to which the metabolite is associated with a particular flora, where * is a significant association and ** is a very significant association. (<a href="#animals-14-02690-f008" class="html-fig">Figure 8</a>a,b).</p>
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20 pages, 4220 KiB  
Review
Upgrading In Vitro Digestion Protocols with Absorption Models
by Otilia Antal, István Dalmadi and Krisztina Takács
Appl. Sci. 2024, 14(18), 8320; https://doi.org/10.3390/app14188320 (registering DOI) - 15 Sep 2024
Viewed by 232
Abstract
Intestinal digestion and absorption are complex processes; thus, it is a challenge to imitate them realistically. There are numerous approaches available, with different disadvantages and advantages. The simplest methods to mimic absorption are the non-cell-based transport models but these lack important characteristics of [...] Read more.
Intestinal digestion and absorption are complex processes; thus, it is a challenge to imitate them realistically. There are numerous approaches available, with different disadvantages and advantages. The simplest methods to mimic absorption are the non-cell-based transport models but these lack important characteristics of enterocytes of the intestine. Therefore, the most often used method is to measure absorption through viable mammalian cells (most commonly Caco-2 cells, cultured on membrane insert plates), which not only assures the incorporation of brush border enzymes (responsible for the final digestion of peptides and disaccharides), it also simulates the absorption process. This means that influx/efflux transporter-facilitated transport, carrier-mediated transport, endocytosis, and transcytosis is also imitated besides passive diffusion. Still, these also lack the complexity of intestinal epithelium. Organoids or ex vivo models are a better approach if we want to attain precision but the highest accuracy can be achieved with microfluidic systems (gut-on-a-chip models). We propose that more research is necessary, and food absorption should also be studied on gut-on-a-chips, especially with fragmented organoids. Our review supports the choices of a proper intestinal epithelium model, which may have a key role in functional food development, nutrition studies, and toxicity assessment. Full article
(This article belongs to the Special Issue Feature Review Papers in Section ‘Food Science and Technology')
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<p>The small intestinal epithelium cells and their localization [<a href="#B6-applsci-14-08320" class="html-bibr">6</a>].</p>
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<p>Transport processes in the intestinal epithelium [<a href="#B21-applsci-14-08320" class="html-bibr">21</a>].</p>
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<p>Approaches for the investigation of the absorption of food components. In Transwell<sup>®</sup> systems, primary cells, cell lines, or fragmented organoids derived from stem cells can be cultured.</p>
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<p>Schematic of transepithelial electrical resistance (TEER) measurements [<a href="#B58-applsci-14-08320" class="html-bibr">58</a>].</p>
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<p>The structure of organoids compared to the intestinal epithelium [<a href="#B64-applsci-14-08320" class="html-bibr">64</a>].</p>
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<p>The best-known method to simulate peristaltic motility in a gut-on-a-chip [<a href="#B42-applsci-14-08320" class="html-bibr">42</a>].</p>
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<p>The schematic organization of the “Intestine Chip” [<a href="#B73-applsci-14-08320" class="html-bibr">73</a>], based on [<a href="#B65-applsci-14-08320" class="html-bibr">65</a>].</p>
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<p>Illustration of the Ussing chamber [<a href="#B47-applsci-14-08320" class="html-bibr">47</a>].</p>
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<p>Illustration of everted gut sac [<a href="#B47-applsci-14-08320" class="html-bibr">47</a>].</p>
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20 pages, 5074 KiB  
Article
Chlorogenic Acid Enhances the Intestinal Health of Weaned Piglets by Inhibiting the TLR4/NF-κB Pathway and Activating the Nrf2 Pathway
by Beibei Zhang, Min Tian, Jing Wu, Yueqin Qiu, Xiaoming Xu, Chaoyang Tian, Jing Hou, Li Wang, Kaiguo Gao, Xuefen Yang and Zongyong Jiang
Int. J. Mol. Sci. 2024, 25(18), 9954; https://doi.org/10.3390/ijms25189954 (registering DOI) - 15 Sep 2024
Viewed by 228
Abstract
Chlorogenic acid (CGA) is a natural polyphenol with potent antioxidant and anti-inflammatory activities. However, the exact role of it in regulating intestinal health under oxidative stress is not fully understood. This study aims to investigate the effects of dietary CGA supplementation on the [...] Read more.
Chlorogenic acid (CGA) is a natural polyphenol with potent antioxidant and anti-inflammatory activities. However, the exact role of it in regulating intestinal health under oxidative stress is not fully understood. This study aims to investigate the effects of dietary CGA supplementation on the intestinal health of weaned piglets under oxidative stress, and to explore its regulatory mechanism. Twenty-four piglets were randomly divided into two groups and fed either a basal diet (CON) or a basal diet supplemented with 200 mg/kg CGA (CGA). CGA reduced the diarrhea rate, increased the villus height in the jejunum, and decreased the crypt depth in the duodenum, jejunum, and ileum of the weaned piglets (p < 0.05). Moreover, CGA increased the protein abundance of Claudin-1, Occludin, and zonula occludens (ZO)-1 in the jejunum and ileum (p < 0.05). In addition, CGA increased the mRNA expression of pBD2 in the jejunum, and pBD1 and pBD2 in the ileum (p < 0.05). The results of 16S rRNA sequencing showed that CGA altered the ileal microbiota composition and increased the relative abundance of Lactobacillus reuteri and Lactobacillus pontis (p < 0.05). Consistently, the findings suggested that the enhancement of the intestinal barrier in piglets was associated with increased concentrations of T-AOC, IL-22, and sIgA in the serum and T-AOC, T-SOD, and sIgA in the jejunum, as well as T-AOC and CAT in the ileum caused by CGA (p < 0.05). Meanwhile, CGA decreased the concentrations of MDA, IL-1β, IL-6, and TNF-α in the serum and jejunum and IL-1β and IL-6 in the ileum (p < 0.05). Importantly, this study found that CGA alleviated intestinal inflammation and oxidative stress in the piglets by inhibiting the TLR4/NF-κB signaling pathway and activating the Nrf2 signaling pathway. These findings showed that CGA enhances the intestinal health of weaned piglets by inhibiting the TLR4/NF-κB pathway and activating the Nrf2 pathway. Full article
(This article belongs to the Special Issue Antibacterial and Antioxidant Effects of Plant-Sourced Compounds)
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<p>Effects of dietary supplementation with CGA on the villi height and crypt depth of the small intestine in piglets. (<b>A</b>) H&amp;E staining of the intestine (scale bar, 500 μm); (<b>B</b>–<b>D</b>) analysis of villi height and crypt depth in the intestine. Values are presented as the mean ± SEM (<span class="html-italic">n</span> = 6). *, 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05. **, <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Effects of dietary CGA supplementation on the expression of tight junctions in the jejunum and ileum. (<b>A</b>,<b>B</b>) The immunoreactivity of tight junctions in the jejunum and ileum of piglets; (<b>C</b>–<b>F</b>) Western blot analysis of tight junctions in the jejunum and ileum of piglets. <span class="html-italic">n</span> = 6. *, 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05. **, <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Effects of dietary CGA supplementation on the mRNA expression of mucins and porcine beta defensins in the jejunum (<b>A</b>) and ileum (<b>B</b>) mucosa of piglets. MUC, mucin; pBD, porcine beta defensins; PG1, Protegrin-1. Values are presented as the mean ± SEM (<span class="html-italic">n</span> = 6). *, 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05.</p>
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<p>Effects of dietary CGA supplementation on the ileal microbiota of piglets. (<b>A</b>–<b>G</b>) Relative abundance of microbiota at the phylum, genus, and species levels. (<b>H</b>) Beta diversity; (<b>I</b>–<b>K</b>) alpha diversity. Values are presented as the mean ± SEM (<span class="html-italic">n</span> = 6). *, 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05. **, <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Effects of dietary supplementation with CGA on antioxidant status and immune-inflammatory level in the serum of piglets. (<b>A</b>–<b>D</b>) The antioxidative and oxidative indicators in the serum. (<b>E</b>–<b>H</b>); the concentration of inflammatory factors in the serum; (<b>I</b>,<b>J</b>) the immunoglobulin concentration in the serum. Values are presented as the mean ± SEM (<span class="html-italic">n</span> = 6). *, 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05. **, <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Effects of dietary supplementation with CGA on antioxidant status and immune-inflammatory level in the jejunum and ileum of piglets. (<b>A</b>–<b>D</b>) The antioxidative and oxidative indicators in the serum; (<b>E</b>–<b>H</b>) the concentration of inflammatory factors in the serum; (<b>I</b>) the immunoglobulin concentration in the serum. Values are presented as the mean ± SEM (<span class="html-italic">n</span> = 6). *, 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05. **, <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Effects of dietary CGA supplementation on the activation of the NF-κB and Nrf2 signaling pathways in the jejunum and ileum of piglets. (<b>A</b>–<b>D</b>) Jejunum; (<b>E</b>–<b>H</b>) ileum. *, 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05. **, <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Correlation analysis between microorganisms, oxidative stress indicators, immunoglobulins, and inflammatory factors in the intestines of piglets. *, 0.01 &lt; <span class="html-italic">p</span> ≤ 0.05. **, <span class="html-italic">p</span> ≤ 0.01.</p>
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<p>Network pharmacological analysis between CGA, oxidative stress, and inflammation. (<b>A</b>,<b>B</b>) Intersection analysis between CGA targets and the disease targets of oxidative stress and inflammation, as well as screening of core targets; (<b>C</b>) the schematic diagram of the interaction between CGA and TLR4; (<b>D</b>) the top 20 KEGG pathways.</p>
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11 pages, 3831 KiB  
Article
A Custom-Developed Device for Testing Tensile Strength and Elasticity of Vascular and Intestinal Tissue Samples for Anastomosis Regeneration Research
by Zoltan Attila Godo, Laszlo Adam Fazekas, Gergo Fritsch, Balazs Szabo and Norbert Nemeth
Sensors 2024, 24(18), 5984; https://doi.org/10.3390/s24185984 - 15 Sep 2024
Viewed by 346
Abstract
Optimizing the regeneration process of surgically created anastomoses (blood vessels, intestines, nerves) is an important topic in surgical research. One of the most interesting parameter groups is related to the biomechanical properties of the anastomoses. Depending on the regeneration process and its influencing [...] Read more.
Optimizing the regeneration process of surgically created anastomoses (blood vessels, intestines, nerves) is an important topic in surgical research. One of the most interesting parameter groups is related to the biomechanical properties of the anastomoses. Depending on the regeneration process and its influencing factors, tensile strength and other biomechanical features may change during the healing process. Related to the optimal specimen size, the range and accuracy of measurements, and applicability, we have developed a custom-tailored microcontroller-based device. In this paper, we describe the hardware and software configuration of the latest version of the device, including experiences and comparative measurements of tensile strength and elasticity of artificial materials and biopreparate tissue samples. The machine we developed was made up of easily obtainable parts and can be easily reproduced on a low budget. The basic device can apply a force of up to 40 newtons, and can grasp a 0.05–1 cm wide, 0.05–1 cm thick tissue. The length of the test piece on the rail should be between 0.3 and 5 cm. Low production cost, ease of use, and detailed data recording make it a useful tool for experimental surgical research. Full article
(This article belongs to the Special Issue Feature Papers in Biosensors Section 2024)
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<p>Representative picture of the structure of the device from different views. A robust frame is required for stable and accurate operation of the instrument.</p>
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<p>Representative picture of the HJJ-001 type grippers while testing a chicken sciatic nerve (biopreparate).</p>
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<p>Representative superimposed tensile strength measurements stress–strain curve of different suture materials: (<b>A</b>): 4/0 absorbable polyglycolide-poly (e-caprolactone) copolymer suture material (Simfra, Kollsut, North Miami Beach, FL, USA) which we usually use for bowel anastomosis; (<b>B</b>): 5/0 non-absorbable silk suture material (Silk, SMI, Vith, Belgium) using for teaching purposes. (n = 5 per group; L<sub>0</sub> = 8 mm; motor speed: 1.95 mm/s).</p>
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<p>Representative tensile strength measurement stress–strain curves of different tissue biopreparates. The different mechanical properties are easily recognizable, even within a single tissue, as separate ruptures of serosa and mucosa layers (motor speed: 1.95 mm/s).</p>
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<p>Analysis of force–elongation (stress–strain) curve. The initial part of the curve (red) was not included in the slope calculation due to its irregularity (femoral artery biopreparate of a rat; L<sub>0</sub> = 8 mm; motor speed: 1.95 mm/s).</p>
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<p>Representative analysis of the exported data. There were some irregularities in the beginning (even some negative values) so after the gram/newton conversion we applied a filter (0.0196–maximum). The entire filtered curves were divided into two parts: the first-third (0–33%) and the remaining two-thirds (34–100%). The slope of these curves was determined using the following formula in Excel = SLOPE(known_y’s, known_x’s) where x = LΔ and y = applied force. The calculation was equal to the slope of the regression line: slope = tgα = <math display="inline"><semantics> <mrow> <mstyle scriptlevel="0" displaystyle="true"> <mfrac> <mrow> <mo>∑</mo> <mfenced separators="|"> <mrow> <mi>x</mi> <mo>−</mo> <mover accent="true"> <mrow> <mi>x</mi> </mrow> <mo>¯</mo> </mover> </mrow> </mfenced> <mo>∗</mo> <mfenced separators="|"> <mrow> <mi>y</mi> <mo>−</mo> <mover accent="true"> <mrow> <mi>y</mi> </mrow> <mo>¯</mo> </mover> </mrow> </mfenced> </mrow> <mrow> <mo>∑</mo> <msup> <mrow> <mfenced separators="|"> <mrow> <mi>x</mi> <mo>−</mo> <mover accent="true"> <mrow> <mi>x</mi> </mrow> <mo>¯</mo> </mover> </mrow> </mfenced> </mrow> <mrow> <mn>2</mn> </mrow> </msup> </mrow> </mfrac> </mstyle> </mrow> </semantics></math>. (5/0 non-absorbable silk suture material (Silk, SMI, Belgium); L<sub>0</sub> = 8 mm; motor speed: 1.95 mm/s.)</p>
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16 pages, 1137 KiB  
Article
Smells Like Anthelmintic Resistance—Gastrointestinal Prevalence, Burden and Diversity in Dogs from Portugal
by Cláudia Luís Batista, Ricardo Cabeças, Cláudio Araújo-Paredes, Maria Aires Pereira and Teresa Letra Mateus
Pathogens 2024, 13(9), 799; https://doi.org/10.3390/pathogens13090799 - 14 Sep 2024
Viewed by 320
Abstract
Anthelmintic resistance has been documented worldwide, but few cases have been reported in dogs. Shelter dogs are a great opportunity to study intestinal helminths and assess their potential resistance to anthelmintic compounds. With these two goals in mind, 361 faecal samples were collected [...] Read more.
Anthelmintic resistance has been documented worldwide, but few cases have been reported in dogs. Shelter dogs are a great opportunity to study intestinal helminths and assess their potential resistance to anthelmintic compounds. With these two goals in mind, 361 faecal samples were collected from dogs in 18 municipalities in Portugal, from 19 shelters and 11 private tutors. These samples were analysed using the Mini-FLOTAC before and 13 days after deworming. The percentage of faecal egg count reduction was calculated. Parasitic forms were identified in 22.4% of the samples collected: Trichuris vulpis (10.5%), Toxocara canis (8.0%), members of the family Ancylostomatidae (6.4%), Toxascaris leonina (0.8%), Dipylidium caninum (0.6%) and members of the family Taeniidae (0.3%). The first three nematode parasites showed the highest intensity of infection (2900, 1325, and 1265 eggs per gram, respectively). In the second faecal collection, parasites were present in 20.8% of the samples. The anthelmintics febendazole + pyrantel embonate + praziquantel and praziquantel + fenbendazole were ineffective for T. vulpis and members of the family Ancylostomatidae in 16.2% and 6.9% of the samples, respectively. The parasites identified are all potentially zoonotic. This study is the first to suggest a potential resistance of T. vulpis to anthelmintics. Full article
(This article belongs to the Section Vaccines and Therapeutic Developments)
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<p>Geographical distribution and diversity of intestinal helminths identified and possible anthelmintic resistance in dogs’ helminths in different municipalities of the north and centre of Portugal.</p>
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23 pages, 731 KiB  
Review
Substitutive Effects of Milk vs. Vegetable Milk on the Human Gut Microbiota and Implications for Human Health
by Alicia del Carmen Mondragon Portocarrero, Aroa Lopez-Santamarina, Patricia Regal Lopez, Israel Samuel Ibarra Ortega, Hatice Duman, Sercan Karav and Jose Manuel Miranda
Nutrients 2024, 16(18), 3108; https://doi.org/10.3390/nu16183108 (registering DOI) - 14 Sep 2024
Viewed by 479
Abstract
Background: In the last two decades, the consumption of plant-based dairy substitutes in place of animal-based milk has increased in different geographic regions of the world. Dairy substitutes of vegetable origin have a quantitative composition of macronutrients such as animal milk, although the [...] Read more.
Background: In the last two decades, the consumption of plant-based dairy substitutes in place of animal-based milk has increased in different geographic regions of the world. Dairy substitutes of vegetable origin have a quantitative composition of macronutrients such as animal milk, although the composition of carbohydrates, proteins and fats, as well as bioactive components, is completely different from that of animal milk. Many milk components have been shown to have relevant effects on the intestinal microbiota. Methods: Therefore, the aim of this review is to compare the effects obtained by previous works on the composition of the gut microbiota after the ingestion of animal milk and/or vegetable beverages. Results: In general, the results obtained in the included studies were very positive for animal milk intake. Thus, we found an increase in gut microbiota richness and diversity, increase in the production of short-chain fatty acids, and beneficial microbes such as Bifidobacterium, lactobacilli, Akkermansia, Lachnospiraceae or Blautia. In other cases, we found a significant decrease in potential harmful bacteria such as Proteobacteria, Erysipelotrichaceae, Desulfovibrionaceae or Clostridium perfingens after animal-origin milk intake. Vegetable beverages have also generally produced positive results in the gut microbiota such as the increase in the relative presence of lactobacilli, Bifidobacterium or Blautia. However, we also found some potential negative results, such as increases in the presence of potential pathogens such as Enterobacteriaceae, Salmonella and Fusobacterium. Conclusions: From the perspective of their effects on the intestinal microbiota, milks of animal origin appear to be more beneficial for human health than their vegetable substitutes. These different effects on the intestinal microbiota should be considered in those cases where the replacement of animal milks by vegetable substitutes is recommended. Full article
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<p>Gut microbiota and its influence in different aspects of human health.</p>
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14 pages, 4957 KiB  
Article
Toxicity Evaluation of Potassium Sorbate In Vivo with Drosophila Melanogaster
by Xubo Zhang, Qian Zhang, Xiaoxuan Song, Wanchen Yang, Andi Cheng, Jianzhen Zhang and Wei Dong
Insects 2024, 15(9), 703; https://doi.org/10.3390/insects15090703 - 14 Sep 2024
Viewed by 327
Abstract
Potassium sorbate (PS) is a preservative widely used in the food, pharmaceutical, and cosmetics industries. Improper and careless use of PS can lead to various health issues and potential environmental problems. Drosophila is capable of making rapid and sensitive responses to stress or [...] Read more.
Potassium sorbate (PS) is a preservative widely used in the food, pharmaceutical, and cosmetics industries. Improper and careless use of PS can lead to various health issues and potential environmental problems. Drosophila is capable of making rapid and sensitive responses to stress or other stimuli. Here we utilized Drosophila as a model organism to evaluate the potential toxicity of PS. Our study revealed that PS ingestion reduced the lifespan and fecundity of Drosophila. In addition, excessive PS ingestion led to cell apoptosis and ROS accumulation in the midgut. Furthermore, PS intake also enhanced the mitophagy of midgut cells. Strikingly, PS affected the cell differentiation progression as well, leading to the production of more enteroendocrine (EE) cells. We further demonstrated that the expression of notch (N), a vital player in intestinal stem cell (ISC) differentiation, was down-regulated in the midgut. This indicates that the differentiation progression was affected potentially by repressing the N expression. Full article
(This article belongs to the Section Insect Physiology, Reproduction and Development)
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<p>The impact of PS intake on the lifespan and fecundity of <span class="html-italic">Drosophila</span>. (<b>A</b>) PS intake led to a reduction in <span class="html-italic">Drosophila</span> longevity. (<b>B</b>) The LT50 of flies fed with PS. (<b>C</b>) The overall quantity of eggs laid over a span of 5 days. <span class="html-italic">p</span> ≤ 0.05 was considered significant (*), <span class="html-italic">p</span> ≤ 0.001 (***), and <span class="html-italic">p</span> ≤ 0.0001 (****).</p>
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<p>PS impacts the cell membrane of the midgut, while not affecting the pH. (<b>A</b>) UAS-CD8-RFP was driven by c601-Gal4 to mark the cell membrane of the midgut. The cell membrane in the midguts of flies fed with 0.1% and 0.5% PS appears fuzzy, specifically in the midguts of 0.5% PS-fed flies. (<b>B</b>) An acidic area, indicated by the yellow color, is noticeable in the center of control midgut, while other regions appeared blue. BPB staining demonstrated that the midgut’s pH was not visibly influenced by 0.1% and 0.5% PS ingestion.</p>
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<p>PS ingestion resulted in cell apoptosis in the midgut. (<b>A</b>) The Trypan blue staining exhibited induced cell death in the midguts fed with 0.1% and 0.5% PS. (<b>B</b>–<b>D</b>) The puc-lacZ staining also demonstrated that cell death was induced by PS ingestion.</p>
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<p>PS ingestion resulted in ROS accumulation in the midgut. (<b>A</b>–<b>C</b>) The figure shows ROS detection in the midguts of flies fed on standard medium or PS medium (0.1% and 0.5%). Scale bar = 50 μm.</p>
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<p>PS ingestion induced mitophagy in the midgut. (<b>A</b>–<b>C</b>) The midguts from flies that were fed on 0.1% and 0.5% PS medium exhibited more severe mitophagy when compared with the control group. White arrows indicate the red-only punctas.</p>
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<p>The gene expression levels in the midguts of flies fed with standard medium or PS medium. (<b>A</b>) The expression levels of genes related to cell membrane integrity. (<b>B</b>) The expression levels of genes related with cell apoptosis or mitophagy. All data are reported as means ± SEM of three independent biological replications. The asterisks indicate significance differences between the control and PS-fed groups (<span class="html-italic">p</span> ≤ 0.01, **), while “ns” means no statistically significant differences.</p>
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<p>PS ingestion alters ISCs differentiation trajectory. (<b>A</b>–<b>C</b>) EE cells in the midgut from flies fed with 0.1% and 0.5% PS medium were labeled by Prospero antibody staining. (<b>D</b>) Quantitative analysis of the Prospero antibody-labeled EE cells, the number of EE cells were increased in the midguts of the flies fed with PS. All data are reported as means ± SEM of three independent biological replications. <span class="html-italic">p</span> ≤ 0.05 was considered significant (*) and <span class="html-italic">p</span> ≤ 0.0001 (****).</p>
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<p>PS ingestion led to the reduction of Notch signaling. (<b>A</b>) The N-GFP in the midgut of flies fed with the standard medium or PS medium (0.1% and 0.5%). Scale bar = 50 μm. (<b>B</b>) PS ingestion did not significantly influence the number of N-GFP clones. (<b>C</b>) PS ingestion notably reduced the size of N-GFP clones. (<b>D</b>) RT-qPCR result showed that the <span class="html-italic">notch</span> expression was down-regulated in midgut of 0.5% PS-fed flies. All data are reported as means ± SEM of three independent biological replications. The asterisks indicate significance differences between the control and PS-fed groups (<span class="html-italic">p</span> ≤ 0.01, **; <span class="html-italic">p</span> ≤ 0.0001 ****), while “ns” means no statistically significant differences.</p>
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15 pages, 851 KiB  
Article
Effects of Cashew Nuts (Anacardium occidentale L.) and Cashew Nut Oil on Intestinal Permeability and Inflammatory Markers during an Energy-Restricted 8-Week Intervention: A Randomized Controlled Trial (Brazilian Nuts Study)
by Talitha Silva Meneguelli, Aline Lage Wendling, Ana Claudia Pelissari Kravchychyn, Daniela Mayumi Usuda Prado Rocha, Ana Paula Dionísio, Josefina Bressan, Hércia Stampini Duarte Martino, Elad Tako and Helen Hermana Miranda Hermsdorff
Foods 2024, 13(18), 2917; https://doi.org/10.3390/foods13182917 - 14 Sep 2024
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Abstract
Cashew nuts can contribute to improving intestinal permeability and inflammation as they contain essential nutrients and bioactive compounds, but no clinical trials have evaluated these potential effects. This randomized trial aimed to assess the effects of cashew nuts and their oil on intestinal [...] Read more.
Cashew nuts can contribute to improving intestinal permeability and inflammation as they contain essential nutrients and bioactive compounds, but no clinical trials have evaluated these potential effects. This randomized trial aimed to assess the effects of cashew nuts and their oil on intestinal permeability and inflammatory markers. Sixty-four adults with overweight or obesity were allocated into three groups receiving energy restriction (−500 kcal/day): control (CT, free nuts), cashew nuts (CN, 30 g/day), or cashew nut oil (OL, 30 mL/day). Urine lactulose and mannitol, plasma zonulin and the lipopolysaccharide-binding protein (LBP), plasma interleukins (IL-6, TNF-α, IL-10, IL-1β, IL-8, and IL-12p70), and C-reactive proteins were analyzed. Energy restriction reduced body fat and other indicators of adiposity without differences between the groups. Only the control group increased LBPs after an 8-week intervention. There were no statistically significant differences found between the groups in terms of intestinal permeability and inflammatory markers. In conclusion, incorporating cashew nuts or cashew nut oil into an energy-restricted 8-week dietary intervention did not change intestinal permeability and inflammatory markers. As studies evaluating cashew nuts on these markers remain scarce, further research is needed, perhaps with a longer study period and a higher concentration of cashew nuts and oil. Full article
(This article belongs to the Section Food Nutrition)
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