Scale-up production of and dietary supplementation with the recombinant antimicrobial peptide tilapia piscidin 4 to improve growth performance in Gallus gallus domesticus.

Antimicrobial peptides (AMPs) are short and positively charged peptides with broad-spectrum antimicrobial activities. AMPs have been investigated as potential antibiotic alternatives to improve growth performance and prevent pathogen infection in the poultry industry. The antimicrobial peptide tilapia piscidin 4 (TP4) was derived from Oreochromis niloticus, possesses antimicrobial activities and immunomodulatory properties, promotes intestinal health, and protects against pathogen infection. The codon-optimized sequence of TP4 was introduced into the pPICZαA vector and transformed into Pichia pastoris. Large-scale expression was induced following culture with methanol in a 500-liter fermenter. Freeze drying of fermented rTP4 broth and then rTP4 evaluation as a feed additive for Gallus gallus domesticus were performed. The in vitro antimicrobial activity of recombinant TP4 (rTP4) against gram-positive and gram-negative pathogens was evaluated. Evaluation of the effect of temperature on the antimicrobial activity of rTP4 showed its high stability at high temperatures. rTP4 significantly enhanced the phagocytic activity of macrophage cells, indicating that rTP4 has a remarkable ability to stimulate macrophages. rTP4 was used as a dietary supplement at 0.75, 1.5, 3.0, 6.0 and 12% in G. g. domesticus for five weeks, and growth performance, gut microbiota composition, and histology were assessed. The 3.0% rTP4 supplement group showed a significant increase in weight gain ratio and feed efficiency compared to those of the basal broiler diet group. Crude rTP4 was expressed by yeast to significantly promote growth efficiency and resistance against pathogens in G. g. domesticus, which could indicate its use as a suitable alternative to antibiotics as feed additives in the poultry industry.

Six weeks of Jilin ginseng root supplementation attenuates drop jump-related muscle injury markers in healthy female college students.

BACKGROUND: Eccentric muscle contraction is an inherent component of numerous sporting movements but can result in muscle fatigue and injury, especially when engaging in unfamiliar exercise, which requires pharmacological intervention. Jilin ginseng root (GS) has been used to protect muscles and reduce the risk of exercise injury. AIM OF THE STUDY: In this study, we sought to examine and demonstrate the effectiveness of using GS in preventing muscle stiffness and reducing the risk of exercise injury in women. METHODS: Twenty females were randomly assigned to GS and placebo groups. Body composition, serum biochemistry index, kinematics, and endurance exercise tests were measured at two time point presupplementation and 6 weeks after supplementation. The major compounds of GS were characterized using a high-performance liquid chromatograph with a gradient delivery system (HPLC). RESULTS: After 6 weeks of supplementation, the GS group exhibited significant increases in the serum levels of free fatty acids and glucose as well as greater maximum oxygen consumption (VO2 max, mL min-1 kg-1) compared with the placebo group in an exhaustive biking test. Following drop jump tests, the jump height and reactive strength index were increased in the GS group after completing 70 DJs. In addition, subjects in the GS group also showed decreased knee and ankle stiffness in DJs, leading to reduced fatigue associated with eccentric movement. CONCLUSIONS: GS supplementation leads to ameliorates drop jump muscle stiffness and fatigue in females and is to be used as a nutrient supplement to reduce the risk of musculoskeletal system injuries when performing drop jumps.

Dual expression of transgenic delta-5 and delta-6 desaturase in tilapia alters gut microbiota and enhances resistance to Vibrio vulnificus infection.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exhibit antibacterial and anti-inflammatory activities. Furthermore, diets rich in n-3 PUFAs are known to improve disease resistance and limit pathogen infection in commercial aquaculture fishes. In this study, we examined the effects of transgenic overexpression of n-3 PUFA biosynthesis genes on the physiological response to bacterial infection in tilapia. We first established tilapia strains with single or dual expression of salmon delta-5 desaturase and/or delta-6 desaturase and then challenged the fish with Vibrio vulnificus infection. Interestingly, our data suggest that n-3 PUFA-mediated alterations in gut microbiota may be important in determining disease outcome via effects on immune response of the host. Both liver- and muscle-specific single and dual expression of delta-5 desaturase and delta-6 desaturase resulted in higher n-3 PUFA content in transgenic fish fed with a LO basal diet. The enrichment of n-3 PUFAs in dual-transgenic fish is likely responsible for their improved survival rate and comparatively reduced expression of inflammation- and immune-associated genes after V. vulnificus infection. Gut microbiome analysis further revealed that dual-transgenic tilapia had high gut microbiota diversity, with low levels of inflammation-associated microbiota (i.e., Prevotellaceae). Thus, our findings indicate that dual expression of transgenic delta-5 and delta-6 desaturase in tilapia enhances disease resistance, an effect that is associated with increased levels of n-3 PUFAs and altered gut microbiota composition.

Conversion and Hydrothermal Decomposition of Major Components of Mint Essential Oil by Small-Scale Subcritical Water Treatment.

Thermal stabilities of four major components (l-menthol, l-menthone, piperitone, and l-menthyl acetate) of Japanese mint essential oil were evaluated via subcritical water treatment. To improve experimental throughput for measuring compound stabilities, a small-scale subcritical water treatment method using ampoule bottles was developed and employed. A mixture of the four major components was treated in subcritical water at 180-240 °C for 5-60 min, and then analyzed by gas chromatography. The results indicated that the order of thermal resistance, from strongest to weakest, was: l-menthyl acetate, l-menthol, piperitone, and l-menthone. In individual treatments of mint flavor components, subsequent conversions of l-menthyl acetate to l-menthol, l-menthol to l-menthone, l-menthone to piperitone, and piperitone to thymol were observed in individual treatments at 240 °C for 60 min. As the mass balance between piperitone and thymol was low, the hydrothermal decomposition of the components was considered to have occurred intensely during, or after the conversion. These results explained the degradation of mint essential oil components under subcritical water conditions and provided the basis for optimizing the extraction conditions of mint essential oils using subcritical water.

Dietary supplementation of recombinant antimicrobial peptide Epinephelus lanceolatus piscidin improves growth performance and immune response in Gallus gallus domesticus.

Supplementing chicken feed with antibiotics can improve survival and prevent disease outbreaks. However, overuse of antibiotics may promote the development of antibiotic-resistant bacteria. Recently, antimicrobial peptides have been proposed as alternatives to antibiotics in animal husbandry. Here, we evaluate the effects of antimicrobial peptide, Epinephelus lanceolatus piscidin (EP), in Gallus gallus domesticus. The gene encoding EP was isolated, sequenced, codon-optimized and cloned into a Pichia pastoris recombinant protein expression system. The expressed recombinant EP (rEP) was then used as a dietary supplement for G. g. domesticus; overall health, growth performance and immunity were assessed. Supernatant from rEP-expressing yeast showed in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, according to an inhibition-zone diameter (mm) assay. Moreover, the antimicrobial peptide function of rEP was temperature independent. The fermentation broth yielded a spray-dried powder formulation containing 262.9 µg EP/g powder, and LC-MS/MS (tandem MS) analysis confirmed that rEP had a molecular weight of 4279 Da, as expected for the 34-amino acid peptide; the DNA sequence of the expression vector was also validated. We then evaluated rEP as a feed additive for G. g. domesticus. Treatment groups included control, basal diet and rEP at different doses (0.75, 1.5, 3.0, 6.0 and 12%). Compared to control, rEP supplementation increased G. g. domesticus weight gain, feed efficiency, IL-10 and IFN-γ production. Our results suggest that crude rEP could provide an alternative to traditional antibiotic feed additives for G. g. domesticus, serving to enhance growth and health of the animals.

Effects of an ethanol extract from Lactobacillus paracasei subsp. paracasei NTU 101 fermented skimmed milk on lipopolysaccharide-induced periodontal inflammation in rats.

The increased incidence of periodontal disease in recent years has garnered considerable attention. Numerous studies have confirmed that probiotics, such as lactic acid bacteria, can ameliorate periodontal inflammation. The current study aimed to assess the effect of an ethanol extract of Lactobacillus paracasei subsp. paracasei NTU 101-fermented skimmed milk (NTU101FM) on lipopolysaccharide (LPS)-induced periodontal inflammation in rats. NTU101FM ethanol extract significantly ameliorated the weight loss caused by periodontal inflammation. NTU101FM ethanol extract treatment also reduced the oral microbial levels and decreased the levels of alveolar bone loss. Finally, NTU101FM ethanol extract was found to ameliorate periodontal inflammation by decreasing the levels of pro-inflammatory cytokines and reducing oxidative stresses induced by LPS. Overall, our findings demonstrate that NTU101FM ethanol extract could be developed as a functional food that could ameliorate periodontal inflammation.

Lactic acid bacteria-fermented product of green tea and Houttuynia cordata leaves exerts anti-adipogenic and anti-obesity effects.

Obesity is associated with higher risks of developing diabetes and cardiovascular disease. Green tea, rich in polyphenolic compounds such as epigallocatechin gallate (EGCG) and epigallocatechin (EGC), has been shown to display anti-obesity effects. Houttuynia cordata leaves have also been shown to exhibit anti-obesity effects due to their chlorogenic acid content. Lactic acid bacteria are able to increase the production of polyphenolic compounds. This study aims to develop a novel anti-obesity fermentation product by combining H. cordata leaf tea with green tea, using Lactobacillus paracasei subsp. paracasei NTU 101 (NTU 101) for fermentation due to the advantages of bioconverting the polyphenolic compounds. The regulation of adipogenesis factors and the anti-obesity effect of the NTU 101-fermented tea were evaluated in an in vitro 3T3-L1 pre-adipocyte model and an in vivo obese rat model, respectively. The results show that the NTU 101-fermented tea, which contained higher EGCG, EGC, and chlorogenic acid levels than unfermented tea, was able to inhibit the lipogenesis of mature 3T3-L1 adipocytes by the stimulation of lipolysis. Furthermore, the body weight gain, body fat pad, and feeding efficiency of obese rats, induced with a high fat diet, were decreased by the oral administration of NTU 101-fermented tea. The significant anti-obesity effect was probably due to lipolysis. However, NTU 101 bacteria cells and EGCG may also act as functional ingredients to contribute to the anti-obesity effects of NTU 101-fermented products.

Analysis of NO-suppressing activity of Strawberry Wine supplemented with ball-milled achenes.

Inflammation is generally thought to be involved in the development of several chronical diseases, therefore, phytochemicals to modulate immune responses has attracted great interests. The objective of the present study was to evaluate the potential anti-inflammatory effects of wine supplemented using ball-milled achene on modulating NO production and inducible nitric oxide synthase (iNOS) expression. Ball-milled achenes were added in strawberry must prior to fermentation, and the wine samples were then concentrated and extracted with water and/or ethanol prior to analysis. Bioactivities of wine extracts were evaluated using the cell viability assay, cell cycle measurements, NO production and iNOS expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Treatments of achenes supplemented strawberry wine extract up to 100 µg/mL inhibited the proliferation of LPS-stimulated RAW264.7 cell via affecting the progression of cell cycle. Moreover, no detectable cytotoxicity in RAW264.7 cells was observed. The supplemented wine extract suppressed the action of LPS and led to a decreased NO production in stimulated cells. The inhibitory effect of the wine extract on NO production was determined to be a 25-40% decrease in the level of 25-100 µg/mL, in contrast to a 10% decrease for conventional wine samples. Additionally, an alcoholic wine extract (100 µg/mL) led to a 40.31% decrease in iNOS expression in LPS-stimulated cells, which was more effective than the same dose of tocopherol. The results show that strawberry wine supplemented with ball-milled achenes causes a substantial inhibition of NO production, and this biofunction is exerted via the down-regulation of iNOS expression.

Effects of equol on deoxycorticosterone acetate salt-induced hypertension and associated vascular dementia in rats.

Oxidative stress is the major cause of neuronal cell degeneration observed in neurodegenerative diseases including vascular dementia (VaD), and hypertension has been found to increase the probability of VaD. Here, we investigated the effects of equol in deoxycorticosterone acetate (DOCA)-salt-induced hypertensive rats (DHRs) and the associated VaD. The systolic blood pressure of rats treated with low- (10 mg per kg body weight) and high-dose (20 mg per kg body weight) equol for 4 weeks was lower than that of the control group by 12.18 and 17.48% in a dose-dependent manner, respectively (p < 0.05), which was regulated by inhibiting angiotensin-converting enzyme (ACE) activity and increasing the nitric oxide (NO) production. Equol-treated DHRs showed a significant decrease in both the swimming distance and time required to reach the escape platform (78.20 to 82.56%, p < 0.05). In addition, the probe trial session and working memory test indicated that equol improved the long- and short-term memory of the rats. Moreover, the brain antioxidant activity was increased by elevating the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) levels, and the malondialdehyde (MDA) content and acetylcholinesterase (AChE) activity were decreased, indicating that equol suppressed oxidative stress. In conclusion, we demonstrated that equol exhibited comparable blood pressure (BP)-lowering and VaD-improving effects with the clinically used drug, lisinopril in DHRs while there was a positive correlation between the doses. Therefore, this bioactive compound may be useful for developing functional foods, thereby extending the application of equol-containing crops.

Lactobacillus plantarum TWK10 Supplementation Improves Exercise Performance and Increases Muscle Mass in Mice.

Lactobacillus plantarum (L. plantarum) is a well-known probiotic among the ingested-microorganism probiotics (i.e., ingested microorganisms associated with beneficial effects for the host). However, few studies have examined the effects of L. plantarum TWK10 (LP10) supplementation on exercise performance, physical fatigue, and gut microbial profile. Male Institute of Cancer Research (ICR) strain mice were divided into three groups (n = 8 per group) for oral administration of LP10 for six weeks at 0, 2.05 × 108, or 1.03 × 109 colony-forming units/kg/day, designated the vehicle, LP10-1X and LP10-5X groups, respectively. LP10 significantly decreased final body weight and increased relative muscle weight (%). LP10 supplementation dose-dependently increased grip strength (p < 0.0001) and endurance swimming time (p < 0.001) and decreased levels of serum lactate (p < 0.0001), ammonia (p < 0.0001), creatine kinase (p = 0.0118), and glucose (p = 0.0151) after acute exercise challenge. The number of type I fibers (slow muscle) in gastrocnemius muscle significantly increased with LP10 treatment. In addition, serum levels of albumin, blood urea nitrogen, creatinine, and triacylglycerol significantly decreased with LP10 treatment. Long-term supplementation with LP10 may increase muscle mass, enhance energy harvesting, and have health-promotion, performance-improvement, and anti-fatigue effects.

Induction of p21(Waf1/Cip1) by garcinol via downregulation of p38-MAPK signaling in p53-independent H1299 lung cancer.

Garcinol, a polyisoprenylated benzophenone, from Garcinia indica fruit rind has possessed anti-inflammatory, antioxidant, antiproliferation, and anticancer activities. However, the anticancer mechanisms of garcinol in lung cancer were still unclear. Therefore, we examine the effects of garcinol on antiproliferation in human lung cancer cells. Treatments with garcinol for 24 h exhibited morphological changes and inhibited the proliferation of H460 (p53-wild type) and H1299 (p53-null) cells in dose- and time-dependent manners. Furthermore, a significant G1 cell cycle arrest was observed in a dose-dependent treatment after H1299 cells were exposed in garcinol, whereas garcinol induced apoptosis rather than cell cycle arrest in H460 cells. Moreover, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin D1, and cyclin D3 were decreased, although cyclin E and cyclin-dependent kinase 6 (CDK6) were increased in garcinol-treated H1299 cells. Meanwhile, the protein levels of CDK inhibitors p21(Waf1/Cip1) and p27(KIP1) also exhibited upregulation after garcinol treatments. The enhanced protein-associated level between p21(Waf1/Cip1) and CDK4/2 rather than p27(KIP1) and CDK4/2 was demonstrated in garcinol-treated cells. Additionally, knock-down p21(Waf1/Cip1) by specific siRNA competently prevented garcinol-induced G1 arrest. Besides, garcinol also inhibited ERK and p38-MAPK activations in time-dependent mode. The pretreatment with p38-MAPK inhibitor but not ERK inhibitor raised garcinol-induced G1 population cells. Co-treatment with p38-MAPK inhibitor and garcinol synergistically elevated cyclin E, p21(Waf1/Cip1), and p27(Kip1) expressions. Meanwhile, overexpression dominant negative p38-MAPK also enhanced garcinol-induced p21(Waf1/Cip1) expression in H1299 cells. Accordingly, our data suggested that garcinol induced G1 cell cycle arrest and apoptosis in lung cancer cells under different p53 statuses. The p53-independent G1 cell cycle arrest induced by garcinol might be through upregulation of p21(Waf1/Cip1) triggered from p38-MAPK signaling inactivation.

Fucoidan supplementation improves exercise performance and exhibits anti-fatigue action in mice.

Fucoidan (FCD) is a well-known bioactive constituent of seaweed extract that possess a wide spectrum of activities in biological systems, including anti-cancer, anti-inflammation and modulation of immune systems. However, evidence on the effects of FCD on exercise performance and physical fatigue is limited. Therefore, we investigated the potential beneficial effects of FCD on ergogenic and anti-fatigue functions following physiological challenge. Male ICR mice from three groups (n = 8 per group) were orally administered FCD for 21 days at 0, 310 and 620 mg/kg/day, which were, respectively, designated the vehicle, FCD-1X and FCD-2X groups. The results indicated that the FCD supplementations increased the grip strength (p = 0.0002) and endurance swimming time (p = 0.0195) in a dose-depend manner. FCD treatments also produced dose-dependent decreases in serum levels of lactate (p < 0.0001) and ammonia (p = 0.0025), and also an increase in glucose level (p < 0.0001) after the 15-min swimming test. In addition, FCD supplementation had few subchronic toxic effects. Therefore, we suggest that long-term supplementation with FCD can have a wide spectrum of bioactivities on health promotion, performance improvement and anti-fatigue.

In vivo hypolipidemic effects and safety of low dosage Monascus powder in a hamster model of hyperlipidemia.

Monascus or more commonly known as red mold rice is fermented rice on which Monascus purpureus has been grown. It has been a traditional Chinese food additive for thousands of years in China. Secondary metabolite product of Monascus, monacolin K, has been proven that it could be used as an antihypercholesterolemic agent. In this study, M. purpureus NTU568 mutated and selected from a monacolin K productivity strain-M. purpureus HM105 produced high quantities of monacolin K at a level of 9,500 mg kg(-1). This research focused on the effect of adding red mold rice powder of M. purpureus NTU568 to a hamster diet on total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol (LDL-C). In the results, the oral administration of Monascus powder in hyperlipidemia hamster was indeed proven to decrease TC, TG, and LDL-C levels. Plasma TC levels in hamster fed with Monascus powder at one-fold dosage [10.78 mg (day 100 g bw)(-1)] for 4 and 8 weeks were significantly lower (31.2 and 22.0%, respectively) than that in hyperlipidemia hamster. Plasma TG (30.1 and 17.9%) and LDL-C levels (36.0 and 20.7%) were also significantly lowered by feeding Monascus powder at one-fold dosage for 4 and 8 weeks compared to hyperlipidemia hamster. In addition, examinations of liver TC and TG levels of hyperlipidemia hamster were also performed and showed similar effects on lipid-lowering action by oral administration of Monascus powder. Since citrinin is a mycotoxin that possesses nephrotoxic and hepatoxic effects, it has a negative impact on the safety of red mold rice for people. This study examined the liver somatic index [plasma glutamyl oxaloacetic transaminase (GOT) and glutamyl pyruvic transaminase (GPT) levels] and liver biopsy to investigate whether Monascus powder induced damage in liver. It was found that the plasma GOT and GPT levels were not significantly increased by feeding Monascus powder. There was no difference in the results of the liver biopsy between the Monascus powder-treated groups and the control group.