Bioactive peptides in the pancreatin-hydrolysates of whey protein support cell proliferation and scavenge reactive oxygen species.

Whey protein (WP) in milk shows physiologically active functions such as cholesterol control and immune system strengthening. In this study, we performed hydrolysis and peptide polarity fractionation to enhance the efficacy and diversity of its physiological activities, using the digesting enzyme, pancreatin. Our results indicate that hydrolysis significantly increased the cell proliferation of the WP fractions, with the lower-polarity fractions showing greater efficacy in this regard. Our results indicate that hydrolysis significantly increases cell proliferation of the WP fractions. Additionally, we confirmed differences in the antioxidant activity of the WP fractions as a function of polarity was confirmed via scavenging 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay in vitro. WP itself did not show anti-inflammatory efficacy. However, all the hydrolyzed fractions downregulated the mRNA expression levels of inflammatory cytokines in all treated cell lines and, based on a senescence-associated (SA)-ß-galactosidase assay, the fraction with the lowest polarity (F6) inhibited cellular senescence to the greatest extent. Furthermore, we identified the peptide sequences with various physiological activities from whey protein hydrolysates through mass spectrometry. Taken together, our results indicate that the fractionation of WP via hydrolysis generates novel functions including promoting cellular cell proliferation, anti-inflammatory effects, and enhancing antioxidant and anti-cellular senescence.

Identification of ß-Glucosidase Activity of Lentilactobacillus buchneri URN103L and Its Potential to Convert Ginsenoside Rb1 from Panax ginseng.

Lentilactobacillus buchneri isolated from Korean fermented plant foods produces ß-glucosidase, which can hydrolyze ginsenoside Rb1 from Panax ginseng to yield ginsenoside Rd. The aim of this study was to determine the mechanisms underlying the extracellular ß-glucosidase activity obtained from Lentilactobacillus buchneri URN103L. Among the 17 types of lactic acid bacteria showing positive ß-glucosidase activity in the esculin iron agar test, only URN103L was found to exhibit high hydrolytic activity on ginsenoside Rb1. The strain showed 99% homology with Lentilactobacillus buchneri NRRLB 30929, whereby it was named Lentilactobacillus buchneri URN103L. Supernatants of selected cultures with ß-glucosidase activity were examined for hydrolysis of the major ginsenoside Rb1 at 40 °C, pH 5.0. Furthermore, the ß-glucosidase activity of this strain showed a distinct ability to hydrolyze major ginsenoside Rb1 into minor ginsenosides Rd and Rg3. Lentilactobacillus buchneri URN103L showed higher leucine arylamidase, valine arylamidase, α-galactosidass, ß-galactosidase, and ß-glucosidase activities than any other strain. We conclude that ß-glucosidase from Lentilactobacillus buchneri URN103L can effectively hydrolyze ginsenoside Rb1 into Rd and Rg3. The converted ginsenoside can be used in functional foods, yogurts, beverage products, cosmetics, and other health products.

Betaine, a component of Lycium chinense, enhances muscular endurance of mice and myogenesis of myoblasts.

Sarcopenia is a disease characterized by the loss of muscle mass and function that occurs mainly in older adults. The present study was designed to investigate the hypothesis that water extract of Lycium chinense (WELC) would improve muscle function and promote myogenesis for sarcopenia. We investigated the effect of water extracts of L. chinense on muscular endurance function and myogenesis to examine its efficacy in sarcopenia. Intake of WELC-containing cheese enhanced the muscular endurance function of mice in treadmill endurance tests. In addition, the cross-sectional areas of muscle fibers in the gastrocnemius muscle of L. chinense-fed mice were greater than that of control mice. Furthermore, WELC and its key component marker substance betaine promoted myogenesis of myoblasts by increasing the expression of the myogenic protein myosin heavy chain 3 (Myh3) and myotube formation. Taken together, our results suggest that L. chinense may potentially be useful in the development of preventive and therapeutic agents for sarcopenia, as well as in providing basic knowledge on myogenesis and muscular functions.

Specific mutations in the genes of MC1R and TYR have an important influence on the determination of pheomelanin pigmentation in Korean native chickens.

OBJECTIVE: The TYR (Tyrosinase) and MC1R (Melanocortin 1 receptor) genes are recognized as important genes involved in plumage pigmentation in Korean native chickens. Specifically, most color patterns in chicken result from differential expression of the TYR gene. In this study, the co-segregation of the pigmentation and sequence of the TYR and MC1R genes was investigated through intercrosses between red (R1q1), red with black and black plumage color types of native Korean chickens. MATERIALS AND METHODS: Using DNA, RNA, and tissue by plumage color of each Korean native chickens, the role of major genes in pigmentation of pheomelanin was evaluated. Reverse transcription polymerase chain reaction, sequencing, western blot, and immunohistochemical were performed to determine the effect of TYR and MC1R genes on plumage pigmentation in Korean native chickens. RESULTS: The KCO line (Korean chicken Ogol: Black-line) with an EEC _ genotype exhibited black feathers, whereas red and red mixed with black chicken with EeC genotype exhibited white feathers. There were notable differences between the base sequences of MC1R and TYR in three Korean chicken breeds, with the highest variation in TYR. Perhaps this is the key characteristics of Korean chicken. Further, we analyzed the expression patterns of MC1R and TYR genes in each type of Korea native chicken and observed that TYR expression was high in feather follicle (R1q2) of KCO tissue. However, native red (Korean chicken red) and native red with black (Korean chicken red dark) chickens have increased TYR expression in the tissue. However, the expression of MC1R was much different from that of TYR. CONCLUSION: In this study, our results suggest that the differences in position and TYR expression levels exert more influence on plumage pigmentation in native Korean chicken breeds than changes in MC1R expression levels.

Effects of Gouda cheese and Allium hookeri on thermogenesis in mice.

Cheese contains various beneficial nutrients, including calcium and whey protein, as well as large amounts of saturated fatty acids. Thus, intake of cheese increases the production of low-density lipoprotein-cholesterol (LDL-C), a well-defined risk factor for cardiovascular disease. Therefore, identification of natural products that inhibit LDL-C production following cheese intake and verification of the efficacy of such products in animal models are essential. Here, we evaluated the effects of Allium hookeri, a well-known traditional herbal remedy, on metabolism and thermogenesis in mice consuming a cheese-containing diet. Intake of A. hookeri extracts significantly blocked increases in body weight and fat mass caused by intake of Gouda cheese in mice. Additionally, increases in blood triglyceride levels following intake of Gouda cheese were alleviated by A. hookeri. Moreover, intake of Gouda cheese enhanced thermogenesis efficiency. Thus, A. hookeri may have applications as an important additive for reducing the risk of metabolic disease resulting from cheese consumption.

Biotransformation of Major Ginsenoside Rb1 to Rd by Dekkera anomala YAE-1 from Mongolian Fermented Milk (Airag).

Dekkera anomala YAE-1 strain separated from "airag" (Mongolian fermented mare's milk) produces ß-glucosidase, which can convert ginsenoside Rb1 from Panax ginseng. Ginseng-derived bioactive components such as ginsenoside Rb1 have various immunological and anticancer activities. Airag was collected from five different mare milk farms located near Ulaanbaatar, Mongolia. YAE-1 strains were isolated from airag to examine the hydrolytic activities of ß-glucosidase on Korean Panax ginseng using an API ZYM kit. Supernatants of selected cultures having ß-glucosidase activity were examined for hydrolysis of the major ginsenoside Rb1 at 40°C, pH 5.0. The YAE-1 strain was found to be nearly identical at 99.9% homology with Dekkera anomala DB-7B, and was thus named Dekkera anomala YAE-1. This strain exerted higher ß-glucosidase activity than other enzymes. Reaction mixtures from Dekkera anomala YAE-1 showed great capacity for converting ginsenoside Rb1 to ginsenoside Rd. The ß-glucosidase produced by Dekkera anomala YAE-1 was able to hydrolyze ginsenoside Rb1 and convert it to Rd during fermentation of the ginseng. The amount of ginsenoside Rd was highly increased from 0 to 1.404 mg/ml in fermented 20% ginseng root at 7 days.

Alpha-Casein and Beta-Lactoglobulin from Cow Milk Exhibit Antioxidant Activity: A Plausible Link to Antiaging Effects.

Studies on the discovery and function of antioxidants are consistently being performed because oxidative stress can cause various diseases. Many compounds and natural products have antioxidant activity in vitro; however, it is often difficult to reproduce their effects in vivo. Additionally, methods to measure antioxidant activities in cells are also scarce. Here, we investigated the antioxidant activity of milk proteins by observing the formation of arsenite-induced stress granules as a tool to evaluate antioxidant activity in cells. Milk proteins not only decreased the formation of stress granules in several cell types but also scavenged 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cations in vitro. In addition, milk proteins inhibited cellular senescence based on an SA-ß-galactosidase assay, and increased differentiation to myotubes from myoblasts isolated from the skeletal muscles of mouse pups. Taken together, our results demonstrate that milk proteins have an antiaging effect, especially prevention of skeletal muscle loss, through their antioxidant activities. PRACTICAL APPLICATION: Our results provide that antioxidant effects of milk proteins containing α-caseins, ß-caseins, and ß-lactoglobulin can mitigate aging-related damage induced by oxidative stress through showing inhibition of cellular senescence and increase of differentiation and maturation of myoblast. Therefore, we suggest that milk proteins could be potent health supplements to prevent aging-associated diseases, especially sarcopenia.

Characterization of Paenibacillus sp. MBT213 Isolated from Raw Milk and Its Ability to Convert Ginsenoside Rb1 into Ginsenoside Rd from Panax ginseng.

This study was conducted to isolate and characterize Paenibacillus sp. MBT213 possessing ß-glucosidase activity from raw milk, and examine the enzymatic capacity on the hydrolysis of a major ginsenoside (Rb1). Strain MBT213 was found to have a high hydrolytic ability on ginsenoside Rb1 by Esculin Iron Agar test. 16S rDNA analysis revealed that MBT213 was Paenibacillu sp. Crude enzyme of MBT213 strain exhibited high conversion capacity on ginsenoside Rb1 into ginsenoside Rd proven by TLC and HPLC analyses. The API ZYM kit confirmed that Paenibacillu sp. MBT213 exerted higher ß-glucosidase and ß-galactosidase activity than other strains. Optimum pH and temperature for crude enzyme were found at 7.0 and 35°C in hydrolysis of ginsenoside Rb1. After 10 d of optimal reaction conditions for the crude enzyme, ginsenoside Rb1 fully converted to ginsenoside Rd. Ginseng roots (20%) were fermented for 14 d, and analyzed by HPLC showed that amount of ginsenoside Rb1 significantly decreased, while that of ginsenoside Rd was significantly increased. The study confirmed that the ß-glucosidase produced by Paenibacillus sp. MBT213 can hydrolyze the major ginsenoside Rb1 and convert to Rd during fermentation of the ginseng. The ß-glucosidase activity of this novel Paenibacillus sp. MBT213 strain may be utilized in development of variety of health foods, dairy foods and pharmaceutical products.

Characteristics of Gouda Cheese Supplemented with Chili Pepper Extract Microcapsules.

In this study, the physicochemical and sensory properties of Gouda cheese supplemented with microcapsules of chili pepper extract were evaluated. Microcapsules of pepper extract were prepared by coacervation technique using gum acacia-gelatin wall and chili pepper oil core. Changes in pH, lactic acid bacteria (LAB) population, and free amino acid (FAA) content after supplementation of Gouda cheese with chili pepper capsules were monitored during ripening. Texture and sensory characteristics of the Gouda cheese ripened for 6 months were evaluated. The supplementation of pepper extract microcapsules (0.5% or 1%, w/w) did not influence the pH values and LAB content of the Gouda cheese (p<0.05) during the ripening period. While the content of total FAA increased with the ripening process in all the cheese groups (p<0.05), no significant difference (p<0.05) in the content of total FAA was observed among the sample groups at each time point. The addition of pepper extract microcapsules (1%, w/w) to Gouda cheese significantly decreased hardness (p<0.05) and negatively affected sensory attributes in terms of taste and texture (p<0.05). The results demonstrated that supplementation with 0.5% pepper extract microcapsules could provide additional bioactive ingredients, along with maintenance of the quality of Gouda cheese.

Thymic Stromal Lymphopoietin Induction Is Mediated by the Major Whey Proteins α-Lactalbumin and ß-Lactoglobulin through the NF-κB Pathway in Immune Cells.

α-Lactalbumin and ß-lactoglobulin are two major whey proteins that specifically bind immunoglobulin E and are suspected as major allergens causing cow's milk allergy (CMA). Recent studies have shown that thymic stromal lymphopoietin is a critical factor linking at the interface of the body and environment to the T-helper 2 response. However, it is not known whether thymic stromal lymphopoietin expression is changed by α-lactalbumin and ß-lactoglobulin in immune cells. Using RT-PCR and ELISA, the present study was conducted to examine if intravenous injection of α-lactalbumin and ß-lactoglobulin increased pro-inflammatory cytokines, T-helper 2 cytokines, and thymic stromal lymphopoietin expression in several immune cells, including macrophages, mast cells, and keratinocytes. Results showed that α-lactalbumin and ß-lactoglobulin induced thymic stromal lymphopoietin, interleukin-6, and tumor necrosis factor-α expression. It was concluded that the allergenicity of α-lactalbumin and ß-lactoglobulin may be attributed to thymic stromal lymphopoietin induction, T-helper 2 cytokines, and pro-inflammatory cytokines.

Bioactive Peptides in Milk and Dairy Products: A Review.

Functionally and physiologically active peptides are produced from several food proteins during gastrointestinal digestion and fermentation of food materials with lactic acid bacteria. Once bioactive peptides (BPs) are liberated, they exhibit a wide variety of physiological functions in the human body such as gastrointestinal, cardiovascular, immune, endocrine, and nervous systems. These functionalities of the peptides in human health and physiology include antihypertensive, antimicrobial, antioxidative, antithrombotic, opioid, anti-appetizing, immunomodulatory and mineral-binding activities. Most of the bioactivities of milk proteins are latent, being absent or incomplete in the original native protein, but full activities are manifested upon proteolytic digestion to release and activate encrypted bioactive peptides from the original protein. Bioactive peptides have been identified within the amino acid sequences of native milk proteins. Due to their physiological and physico-chemical versatility, milk peptides are regarded as greatly important components for health promoting foods or pharmaceutical applications. Milk and colostrum of bovine and other dairy species are considered as the most important source of natural bioactive components. Over the past a few decades, major advances and developments have been achieved on the science, technology and commercial applications of bioactive components which are present naturally in the milk. Although the majority of published works are associated with the search of bioactive peptides in bovine milk samples, some of them are involved in the investigation of ovine or caprine milk. The advent of functional foods has been facilitated by increasing scientific knowledge about the metabolic and genomic effects of diet and specific dietary components on human health.

Effects of Psychrotrophic Bacteria, Serratia liquefaciens and Acinetobacter genomospecies 10 on Yogurt Quality.

The aim of this study was to evaluate the effect of proteolytic (Serratia liquefaciens, match %: 99.39) or lipolytic (Acinetobacter genomospecies 10, match %: 99.90) psychrotrophic bacteria (bacterial counts, analysis of free fatty acids (FFA) and analysis of free amino acids) on the microbial and chemical properties (yogurt composition), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of yogurt during storage. Yogurts were prepared with raw milk preinoculated with each psychrotrophic bacteria. The total solid, fat, and protein content were not affected by preinoculation, but the pH of yogurt preinoculated with psychrotrophic bacteria was higher than in control. There was a dramatic increase in short chain free fatty acids among FFA in yogurt with Acinetobacter genomospecies 10. For 14 d of cold storage condition, SCFFA was 25.3 mg/kg to 34.4 mg/kg (1.36 times increased), MCFFA was 20.4 mg/kg to 25.7 mg/kg (1.26 times increased), and LCFFA was 240.2 mg/kg to 322.8 mg/kg (1.34 times increased). Serratia liquefaciens (match %: 99.39) in yogurt caused a greater accumulation of free amino acids (FAA), especially bitter peptides such as leucine, valine, arginine, and tyrosine, but SDS-PAGE showed that the inoculation of Serratia liquefaciens did not affect the degree of casein degradation during storage. Taken together, the excessive peptides and FFA in yogurt generated from psychrotrophic bacteria could develop off-flavors that degrade the quality of commercial yogurt products.

Purification, characterization, and properties of an alkaline protease produced by Serratia marcescens S3-R1 inhabiting Korean ginseng rhizosphere.

BACKGROUND: An alkaline protease produced by the Serratia marcescens S3-R1 which inhabits in the Korean ginseng rhizosphere was investigated. The purposes of this study were to characterize and purify the bacterial enzyme by four different purification steps: precipitation of enzyme fraction by ammonium sulfate, loading the enzyme pellets on a DEAE-Sepharose anion-exchange chromatograph, separation of the fraction containing enzyme activity by fast protein liquid Mono Q chromatography and identification of the single-band fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then quantification of the single-band fraction by reverse-phase high-performance liquid chromatography. RESULTS: The molecular weight of the purified protease was estimated as 50 308 Da by matrix-assisted laser desorption ionization time-of-flight analysis. The N-terminal amino acid sequence of the protease was identified as Ala-Val-Thr-Ile-Glu-Asp-Ala-Val-Asp-Asp, and the enzyme belongs to the metalloprotease family. The optimal activities of the protease occurred at pH 7-9 and a temperature 40 °C. The ranges of pH and thermal stability of the enzyme were at 7-10 and 30-40 °C, respectively. CONCLUSION: The alkaline protease was successfully purified and characterized from the bacterium Serratia marcescens S3-R1, which has potential for industrial application, including milk protein hydrolysates.

Saponins, especially platycodin D, from Platycodon grandiflorum modulate hepatic lipogenesis in high-fat diet-fed rats and high glucose-exposed HepG2 cells.

AMP-activated protein kinase (AMPK) plays a central role in controlling hepatic lipid metabolism through modulating the downstream acetyl CoA carboxylase (ACC) and sterol regulatory element-binding protein-1c (SREBP-1c) pathway. Saponins, particularly platycodin D, from the roots of Platycodon grandiflorum (Changkil saponins, CKS) have a variety of pharmacological properties, including antioxidant and hepatoprotective properties. The aim of this study was to investigate the effects of CKS on hepatic lipogenesis and on the expression of genes involved in lipogenesis, and the mechanisms involved. CKS attenuated fat accumulation and the induction of the lipogenic genes encoding SREBP-1c and fatty acid synthase in the livers of HFD-fed rats and in steatotic HepG2 cells. Blood biochemical analyses and histopathological examinations showed that CKS prevented liver injury. CKS and platycodin D each increased the phosphorylation of AMPK and acetyl-CoA carboxylase in HFD-fed rats and HepG2 cells. The use of specific inhibitors showed that platycodin D activated AMPK via SIRT1/CaMKKß in HepG2 cells. This study demonstrates that CKS or platycodin D alone can regulate hepatic lipogenesis via an AMPK-dependent signalling pathway.

Overexpression and clinical significance of carcinoembryonic antigen-related cell adhesion molecule 6 in colorectal cancer.

BACKGROUND: Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) inhibits anoikis and affects the malignant phenotype of cancer cells. In this study, we analyzed CEACAM6 as a gene that is highly upregulated in colon cancer tissues, and examined the assertion that CEACAM6 might be a suitable candidate tumor marker for the diagnosis of colon cancer. METHODS: CEACAM6 gene expression in human colon tissues was performed by tissue microarray and analyzed using RT-PCR (each of normal and tumor tissue, n=40) and immunohistochemical and clinicopathological (colon cancer patients, n=143) analyses. RESULTS: CEACAM6 transcriptional and translational levels were significantly upregulated in human tumor tissues compared to non-tumor regions, and clinicopathological analysis revealed a significant correlation between CEACAM6 protein expression and Dukes' stage (p<0.001). High expression levels of CEACAM6 were significantly associated with lower overall survival (p<0.001) and shorter recurrence-free survival (p<0.001). We demonstrated that knockdown of CEACAM6 with CEACAM6-specific small interfering RNA in colorectal cancer cells attenuated invasivity (35%); conversely, the overexpression of CEACAM6 increased invasiveness. CONCLUSIONS: CEACAM6 is significantly upregulated in colon cancer tissues and is closely associated with poor prognosis, indicating that CEACAM6 might be used as a tumor biomarker and a potential therapeutic target for colon cancer.

Upregulation and secretion of macrophage inhibitory cytokine-1 (MIC-1) in gastric cancers.

BACKGROUND: Macrophage inhibitory cytokine-1 (MIC-1), a distant member of the transforming growth factor (TGF)-beta superfamily, has been reported to be upregulated and secreted from several cancers. We examined MIC-1 expression and secretion in gastric cancers. METHODS: MIC-1 mRNA and protein levels in cancer tissues and cell lines were analyzed by RT-PCR and Western blot. MIC-1 expression in cancer tissues and its secretion in serum were analyzed using immunohistochemistry and ELISA. RESULTS: MIC-1 was significantly upregulated in gastric cancer tissues and cell lines. MIC-1 was secreted from gastric SNU620 cells and its levels in the serum of cancer patients were 10-fold higher than those of healthy controls. In addition, the staining of MIC-1 expression was strongly increased in metastatic gastric cancers. CONCLUSIONS: MIC-1 was obviously overexpressed in gastric cancers and MIC-1 secretion into blood may be useful for the prediction of gastric cancer progression.