Improvement of sleep duration and quality through GABAA receptor by whey protein hydrolysate containing DIQK as the active main compound.

This study characterized the sleep activity, sleep mechanism, and active peptides of whey protein hydrolysates selected through behavioral analysis of fruit-flies (Drosophila melanogaster). Sleep-inducing whey protein (WP) hydrolysate was selected through fruit fly behavior analysis, and sleep activity was measured using a pentobarbital model and electroencephalographic analysis. The mechanism of action was confirmed using a γ-aminobutyric acid (GABA) receptor antagonist, and the active peptide was identified using liquid chromatography-mass spectroscopy. Whey protein hydrolysate, prepared using Alcalase and Prozyme (WP-AP), increased sleep time in a dose-dependent manner. WP-AP significantly increased not only sleep time but also slow-wave sleep and showed an insomnia-alleviating effect in a caffeine-induced insomnia mouse model. In addition, the gene and protein expression levels of GABA sub-type A (GABAA) receptors increased in the brains of mice orally administered with WP-AP. Through peptide analysis, the mixture of DIQK, VPPF peptide, and GABA contained in WP-AP was estimated to exhibit sleep activity, and due to its high content, DIQK was speculated to be the main sleep -inducing ingredient. These results indicate that WP-AP has the potential to be used as a new ingredient to improve sleep quality.

Characteristics and Absorption Rate of Whey Protein Hydrolysates Prepared Using Flavourzyme after Treatment with Alcalase and Protamex.

The purpose of this study was to evaluate the physicochemical properties of whey protein hydrolysate and determine changes in absorption rate due to enzymatic hydrolysis. The molecular weight distribution analysis of whey protein concentrate (WPC) and low-molecule whey protein hydrolysate (LMWPH) using the Superdex G-75 column revealed that LMWPH is composed of peptides smaller than those in WPC. Fourier-transform infrared spectroscopy indicated differences in peak positions between WPC and LMWPH, suggesting hydrolysis-mediated changes in secondary structures. Moreover, LMWPH exhibited higher thermal stability and faster intestinal permeation than WPC. Additionally, oral LMWPH administration increased serum protein content at 20 min, whereas WPC gradually increased serum protein content after 40 min. Although the total amount of WPC and LMWPH absorption was similar, LMWPH absorption rate was higher. Collectively, LMWPH, a hydrolysate of WPC, has distinct physicochemical properties and enhanced absorptive characteristics. Taken together, LMWPH is composed of low-molecular-weight peptides with low antigenicity and has improved absorption compared to WPC. Therefore, LMWPH can be used as a protein source with high bioavailability in the development of functional materials.

Characterization of a novel sulfated-rhamnoglucuronan isolated from Korean seaweed Ulva pertusa and its efficacy for treatment of inflammatory bowel disease in mice.

This study aimed to isolate Ulva pertusa polysaccharide (UPP), which elicits anti-inflammatory bowel disease (IBD) effects, from the Korea seaweed U. pertusa and identify its structure. Firstly, UPP was isolated from U. pertusa using hydrothermal extraction and ethanol precipitation. UPP is a novel polysaccharide that exhibits unique structural features such as 3-sulfated rhamnose, glucuronic acid, iduronic acid, and 3-sulfated xylose, which are repeated in 1,4-glycosidic bonds. Prophylactic oral administration of UPP in mice with dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) suppressed the levels of inflammatory cytokines and MAPK- and NF-κB-related factors in the serum and colon tissue. Tight junction (TJ)-related factors such as occludin, claudin-1, and mucin were effectively augmented by UPP in the colon tissue. In addition, UPP administration prevented the DSS treatment-led cecal short chain fatty acid imbalance, and this effect was most evident for propionic acid. In conclusion, UPP isolated from the Korean U. pertusa demonstrates potent anti-IBD activity. Characterization of this ulvan revealed its unique structure. Moreover, its efficacy may be associated with its anti-inflammatory effects and regulation of gut microbiota and TJ proteins. Thus, this study provides new insights into the biological effects of UPP in IBD.

Lactobacillus brevis-Fermented Gamma-Aminobutyric Acid Ameliorates Depression- and Anxiety-Like Behaviors by Activating the Brain-Derived Neurotrophic Factor-Tropomyosin Receptor Kinase B Signaling Pathway in BALB/C Mice.

This study investigated the effects of Lactobacillus brevis-fermented gamma-aminobutyric acid (LB-GABA) on depressive and anxiety-like behaviors with the underlying molecular mechanism in a chronic stress model of BALB/c mice. LB-GABA attenuates both neuronal cell death and the increase of monoamine oxidase activity induced by hydrogen peroxide. Behavioral tests revealed that GABA significantly increased sucrose preference and reduced immobility time in both tail suspension and forced swimming tests. LB-GABA increased exploration of the open arms in the elevated plus maze and restored activity in the open field. Moreover, LB-GABA lowered stress hormone and inflammatory mediator levels. Mechanistically, LB-GABA increased protein levels of BDNF and TrkB, activating downstream targets (AKT, ERK, and CREB), crucial for neuronal survival and plasticity. Furthermore, LB-GABA protected hippocampal neurons from stress-induced cell death and increased serotonin and dopamine levels. Overall, LB-GABA has the potential to alleviate stress-induced depression and anxiety-like symptoms and neuroinflammation by activating the BDNF-TrkB signaling pathway.

Improved digestibility and bioavailability of pea protein following enzymatic treatment and fermentation by lactic acid bacteria.

Peas (Pisum sativum L.) have a potential commercial value because of their high protein content, non-GMO status, low allergenicity. This study examined changes in the digestion and absorption properties of enzyme-modified and fermented pea protein (EFPP) produced through enzymatic treatment of pea protein and fermentation with lactic acid bacteria. EFPP was digested 22.50% more than isolated pea protein (IPP) during the first 15 min in vitro digestion model. Based on the Caco-2 cell monolayer model, EFPP showed 38.40% higher bioavailability than IPP. Gel Permeation Chromatography showed that the average molecular weight of EFPP was 98.79% lower than that of IPP. The secondary structure of protein was analyzed by Fourier transform infrared spectrometry and it showed a 53.92% reduction in ß-sheet of EFPP than IPP. These results indicate that enzyme treatment and fermentation of pea protein reduced the molecular weight, modified the secondary structure, and improved digestibility and bioavailability.

Efficacy and safety of galacto-oligosaccharide in the treatment of functional constipation: randomized clinical trial.

The efficacy and safety of galacto-oligosaccharides (GOS) in treating functional constipation were evaluated in a four-week randomized, double-blind clinical trial on 63 patients who met Rome IV criteria (34 GOS, 29 placebo group). The number of bowel movements per day and changes in the shape of bowel movements in the treatment group significantly improved compared to those in the control group after four weeks. The Patient Assessment Constipation Quality of Life questionnaire showed that satisfaction with constipation significantly increased in the treatment group. The levels of Bifidobacterium sp. and Lactobacillus sp. significantly increased after four weeks of GOS treatment compared to those measured at baseline. No significant adverse drug reactions were identified in any indicator except for pulse rate. Thus, the prebiotic GOS can be safely used in foods and pharmaceuticals to alleviate symptoms of functional constipation by improving the intestinal flora.

Immunostimulating effects of ulvan type polysaccharide isolated from Korean Ulva pertusa in cyclophosphamide-induced immunosuppressed BALB/c mice.

This study aimed to determine the immunostimulatory activities of ulvan type polysaccharides isolated from Ulva pertusa. First, U. pertusa polysaccharide (UPP) mainly consists of rhamnose, glucuronic acid, iduronic acid, and xylose, which are typical ulvan type monosaccharides. UPP induced phosphorylation of the mitogen-activated protein kinase and nuclear factor-kappa B pathways in macrophages, subsequently triggering cytokine release and phagocytosis. The effects were closely associated with pattern recognition receptors such as dectin-1, mannose receptor, CD11b, CD14, and Toll-like receptors 2 and 4. Moreover, prophylactic administration of UPP was found to protect against body weight loss and lymphatic organ damage in cyclophosphamide-induced immunosuppressed mice. In addition, UPP demonstrated significant stimulatory effects on various immunocytes, such as T cells, B cells, macrophages, and natural killer cells derived from the spleen. These effects were closely related to the mitogen-activated protein kinase and nuclear factor-kappa B pathways, and significant secretion of immunostimulatory cytokines such as IL-6, -12, and TNF-α was noted in both blood and spleen samples. Impairment of the short-chain fatty acid balance in the cecum was prevented by UPP administration in a dose-dependent manner. Consequently, these results suggest that the UPP isolated from U. pertusa contributes to immune system activation.

Effect of rhamnogalacturonan-I-rich polysaccharides isolated from crabapple hydrolysates on IL-1ß-induced inflammation in intestinal epithelial cells.

This study aimed to investigate the structural characteristics and intracellular mechanisms of polysaccharides (MP-PE-I) purified from a crabapple (Malus prunifolia) enzymatic hydrolysate (MP-PE). Activity-guided fractionation revealed that MP-PE-I was the active moiety and significantly reduced the production and gene expression of pro-inflammatory factors in interleukin (IL)-1ß-treated intestinal epithelial cells (Caco-2). Moreover, MP-PE-I downregulated the phosphorylation and nuclear localization of proteins involved in the mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways, as evidenced by immunoblotting and immunofluorescence analysis. In antagonistic studies with specific inhibitors of the MAPK and NF-κB pathways, IL-6 inhibition was significantly regulated by p38; IL-8 by IκBα, JNK, and p38; and monocyte chemoattractant protein-1 (MCP-1) by JNK, p38, and ERK. Additionally, MP-PE-I significantly decreased the mRNA and protein expression of IL-1 receptor type 1. Chemical and structural characteristic analyses showed that MP-PE-I is a polysaccharide rich in rhamnogalacturonan (RG)-I and plays a crucial role in intestinal immunomodulation. To our knowledge, this is the first study to demonstrate the intestinal immunomodulatory activity, intracellular mechanisms, and structural characteristics of RG-I-rich polysaccharides isolated from crabapples.

Lactobacillus brevis M2-Fermented Whey Protein Hydrolysate Increases Slow-Wave Sleep via GABAA Receptors in Rodent Models.

In this study, we investigated the effects of whey protein hydrolysate (WPH) fermented with Lactobacillus brevis on sleep behavior and GABAergic mechanisms in rodent models. Fermentation converted the glutamate in WPH to high (3.15 ± 0.21 mg/mL) levels of γ-aminobutyric acid (GABA). Fermented WPH (WP-SF) enhanced sleep duration in mice by increasing GABA content in the brain. The increase in sleep duration induced by WP-SF resulted from an increase in delta wave activity during non-rapid eye movement sleep, and its sleep-promoting effect in a caffeine-induced insomnia model was characterized by an increase in delta waves. WP-SF increased GABAergic receptors at both mRNA and protein levels. Cotreatment with GABAA receptor antagonists abolished the sleep-promoting effects of WP-SF, indicating that WP-SF shares binding sites with antagonists on GABAA receptors. Collectively, WP-SF effectively increased sleep duration by enhancing delta wave activity through GABAergic activation; thus, it is suggested as a functional food-grade ingredient for promoting sleep.

Lactitol Alleviates Loperamide-Induced Constipation in Sprague Dawley Rats by Regulating Serotonin, Short-Chain Fatty Acids, and Gut Microbiota.

The objective of this study was to examine the impact of lactitol on constipation caused by loperamide in Sprague Dawley rats, with a particular emphasis on its underlying mechanisms and potential health advantages. The lactitol effectively improved fecal parameters, intestinal tissue structure, and the expression of constipation-related gene expression and proteins. Lactitol alleviated fecal weight and water content altered by loperamide and enhanced gastrointestinal transit. The administration also restored mucosal and muscular layer thickness. Mechanistically, lactitol upregulated the mRNA expression and/or protein levels of mucins (MUC2 and MUC4), occludin, claudin-1, and zonula occludens, indicating improved intestinal barrier function. Lactitol positively regulated the composition of cecal microbiota, leading to an increased relative abundance of Bifidobacterium, Lactobacillus, and Romboutsia. Conversely, lactitol decreased the relative abundance of Prevotella, Aerococcus, Muribaculum, Blautia, and Ruminococcus. This study demonstrated the potential of lactitol to relieve constipation by modulating the gut microbiota. These findings suggest that lactitol is an alternative to traditional laxatives and has potential as a health-promoting food sweetener.

Protective Effects of Whey Protein Hydrolysate, Treadmill Exercise, and Their Combination against Scopolamine-Induced Cognitive Deficit in Mice.

In this study, the potential of whey protein hydrolysate (WPH) and treadmill exercise to prevent cognitive decline was investigated, along with their neuroprotective mechanisms. Cognitive dysfunction was induced in mice with 1 mg/kg of scopolamine, followed by the administration of WPH at 100 and 200 mg/kg and/or treadmill exercise at 15 m/min for 30 min five days per week. Both WPH administration and treadmill exercise significantly improved the memory of mice with scopolamine-induced cognitive impairment, which was attributed to several key mechanisms, including a reduction in oxidative stress based on decreased levels of reactive oxygen species and malondialdehyde in the brain tissue and an increase in acetylcholine by increasing choline acyltransferase and decreasing acetylcholine esterase levels. Exercise and WPH also exerted neuroprotective effects by inhibiting the hyperphosphorylation of tau proteins, enhancing the expression of the brain-derived neurotrophic factor, and inhibiting apoptosis by reducing the Bax/Bcl2 ratio in conjunction with the downregulation of the mitogen-activated protein kinase pathway. Moreover, the impact of WPH and treadmill exercise extended to the gut microbiome, suggesting a potential link with cognitive improvement. These findings suggest that both WPH intake and treadmill exercise are effective strategies for mitigating cognitive impairment, providing promising avenues for treating neurodegenerative diseases.

Coadministration of Lactulose with Probiotics Ameliorates Loperamide-Induced Constipation in Mice.

We evaluated the efficacy of mixtures of lactulose with probiotic strains to ameliorate constipation and to identify suitable probiotic strains. Constipation was induced in Institute of Cancer Research mice (6-week-old, male) by the administering loperamide (5 mg/kg, twice a day) orally for 5 days, whereas the control group was not treated. To evaluate the laxative effects of the lactulose-probiotic and lactulose-magnesium hydroxide mixtures, fecal parameters, the gastrointestinal (GI) transit ratio, and fecal short-chain fatty acid (SCFA) content were analyzed. The administration of lactulose and Bacillus licheniformis or Saccharomyces boulardii significantly improved stool number and water content, which were reduced by loperamide. The GI transit ratio was significantly increased compared with that of the control group. The combined administration of lactulose and probiotics (B. licheniformis or S. boulardii) increased total SCFA content, including that of acetate, more effectively compared with lactulose alone. Similarly, coadministration of lactulose and magnesium hydroxide improved the loperamide-induced changes in fecal parameters and GI transit as well as increased total SCFA content. Overall, the combination of lactulose and probiotics relieves the symptoms of constipation by increasing SCFA content and is more effective compared with lactulose alone.

Combined Effects of Ziziphus jujuba, Dimocarpus longan, and Lactuca sativa on Sleep-Related Behaviors through GABAergic Signaling.

We aimed to analyze the increase in the sleep-promoting effects based on the mixed ratio of botanical extracts, Ziziphus jujuba seeds, Dimocarpus longan fruits, and Lactuca sativa leaves, using animal models. Behavioral analyses, including an analysis of the total sleep time of Drosophila melanogaster, were conducted to select the optimal mixed ratio of the three botanical extracts. The effects were verified in a caffeine-induced sleepless model, specific neurotransmitter receptor antagonists, and ICR mice. In D. melanogaster exposed to 2.0% of each extract, group behavior was significantly reduced, and the mixed extracts of Z. jujuba, D. longan, and L. sativa (4:1:1 and 1:4:1) significantly increased the total sleep time with individual fruit flies. In the caffeine-induced insomnia model, mixed extracts (4:1:1 and 1:4:1) led to the highest increase in total sleep time. An analysis of locomotor ability revealed a significant reduction in the mobility percentage in the mixed extract groups (0:0:1, 1:0:1, 1:1:1, 4:1:1, and 1:4:1). The administration of Z. jujuba extract and mixed extracts (4:1:1) significantly increased the expression of GABAA-R, whereas the administration of the mixed extracts (4:1:1) and (1:4:1) significantly increased the expression of GABAB-R1 and GABAB-R2, respectively. D. longan extract and the mixed ratio (1:4:1) reduced the subjective nighttime movement and increased the total sleep time in the presence of flumazenil. An analysis of ICR mice indicated that the administration of mixed extracts (4:1:1) significantly increased sleep duration in a dose-dependent manner. These results indicated that the mixed ratio of Z. jujuba, D. longan, and L. sativa extracts, particularly the mixed ratio of 4:1:1, may have sleep-enhancing effects in fruit flies and mice. The study also identified changes in gene expression related to GABA receptors, indicating the potential mechanism for the observed sleep-promoting effects.