Immunomodulatory activities of Lactobacillus rhamnosus ZDY114 and donkey milk in BALB/c mice

The immunomodulatory activity of Lactobacillus rhamnosus ZDY114 and donkey milk in BALB/c mice was evaluated by assessing the splenic lymphocyte transformation, haemolytic complement activity, carbon clearance ability and natural killer cell activity. Results showed donkey milk (5 g kg⁻¹) in combination with L. rhamnosus ZDY114 (5 × 10⁷ cfu mL⁻¹) exhibited a significant increase in splenic lymphocyte proliferation, carbon granule engulfing ability and natural killer cell activity when compared with donkey milk or L. rhamnosus ZDY114 alone (p < 0.05). An elevated response in serum haemolytic activity was only observed when compared with L. rhamnosus ZDY114. In conclusion, donkey milk (5 g kg⁻¹) in combination with L. rhamnosus ZDY114 (5 × 10⁷ cfu mL⁻¹) was able to enhance specific immune functions.     

Induction of the cell cycle arrest and apoptosis by flavonoids isolated from Korean Citrus aurantium L. in non-small-cell lung cancer cells

This study investigated the anti-proliferative and apoptotic effect of flavonoids isolated from Korean Citrus aurantium L. using A549 lung cancer cells. Flavonoids potently inhibited of A549 cells in a dose-dependent manner, whereas flavonoids had a weak inhibitory effect on proliferation of WI-38 cells. Flow cytometry and Western blot analysis showed that flavonoids induced cell cycle arrest at the G2/M checkpoint by controlling the proteins expression level of cyclin B1, cdc2, cdc25c and p21^WAF1/CIP1. Also, flavonoids induced apoptosis through the regulation of the expression of caspases, cleaved PARP and Bax/Bcl-xL ratio. The activity of caspase-3 on A549 cells increased in a dose-dependent manner. These results clearly indicated that the anti-cancer effect of flavonoids on A549 cells follows multiple cellular pathways through G2/M arrest and the induction of apoptosis.     

Japanese apricot extract attenuates nuclear factor-κB activation and proteasomal activity in A549 lung cancer cells

The present study investigated the mechanism of Japanese apricot extract (JAE) in inhibiting lung cancer cells proliferation. JAE inhibited A549 lung cancer cell proliferation at non-cytotoxic doses and suppressed nuclear factor-κB (NF-κB) activation induced by TNF-α at a dose of 1 mg/mL (p<0.05). Proteasome activities of A549 cells were blocked by JAE in a dose-dependent manner at concentrations of 0.67 and 1 mg/mL (p<0.05). These results indicate that JAE has anti-proliferative activity against A549 cells and suppresses NF-κB activation, partially due to inhibiting proteasome activity.     

Antiviral activity of donkey milk protein fractions on echovirus type 5

Antiviral activity of Ragusano donkeys' milk proteins was investigated for the effect on echovirus type 5, known to infect the gastrointestinal tract of humans. Three protein fractions were tested; casein (CN), whey protein (WP) and a low molecular whey protein fraction (LWP; <30,000 Da). The antiviral activity of WP and LWP was tested on echovirus type 5 at three concentrations (1, 5 and 10 mg mL^?1); CN was assessed only at the lower concentration. All donkey milk protein fractions showed significant inhibition on virus replication at the concentration of 1 mg mL^?1, and both WP and LWP fractions showed significant inhibition on the virus replication at all concentrations tested. The strongest antiviral effect was observed for the WP fraction. These findings show that the different whey proteins in donkey milk, probably acting in synergy, exert antiviral activity on echovirus 5 and might contribute to prevent gastrointestinal virus infections in humans.     

Effects of different genotypes at the CSN3 and LGB loci on milk and cheese-making characteristics of the bovine Cinisara breed

The Cinisara is an autochthonous Sicilian bovine breed reared mainly for production of Caciocavallo Palermitano, a typical stretched-curd cheese. The distribution of A and B alleles at both the β-lactoglobulin (LGB) and κ-casein (CSN3) loci in Cinisara cows and their influence on milk traits and cheese yield and composition were analysed. The LGB alleles are associated with significantly different effects on whey protein level (lower for BB genotype, P < 0.01), and casein index (higher value for BB genotype, P < 0.001), while CSN3 alleles were associated with significantly different effects on milk yield and coagulation properties; the BB genotype showed higher values for milk yield (P < 0.01) and curd firmness (a₃₀ P < 0.01 and a_2r P < 0.01) and lower values for coagulation and curd firming time (P < 0.01) than the AA genotype. Cheese made with LGB BB milk showed higher percentage protein recovery (P < 0.01); cheese made with CSN3 BB milk showed higher percentage fat recovery (P < 0.05).     

Khz (fusion of ganoderma lucidum and Polyporus umbellatus mycelia) induces apoptosis in A549 human lung cancer cells by generating reactive oxygen species and decreasing the mitochondrial membrane potential

Khz, a naturally occurring compound derived from the fusion of Ganoderma lucidum and Polyporus umbellatus mycelia, inhibits the growth of cancer cells. This study aimed to investigate the anti-proliferative effects of Khz on A549 lung cancer cells. Khz cytotoxicity was measured using an MTT assay and the mitochondrial membrane potential (MMP)-related, calcium-induced generation of reactive oxygen species (ROS) in A549 cells was measured by flow cytometry. The expression of p53, Bax, Bcl-2, and actin proteins was analyzed by western blotting. Khz inhibited cell division and induced apoptosis in A549 cells. Flow cytometry analysis showed that the percentage of A549 cells in sub-G1 phase of the cell cycle increased in response to Khz treatment. Khz reduced MMP and Bcl-2 protein levels, but increased ROS generation and p53 and pro-apoptotic proteins. The anti-proliferative and pro-apoptotic effects of Khz suggest that this extract shows great promise as a potential chemotherapeutic agent.     

Biochemical changes to milk following treatment by a novel,cold atmospheric plasma system

It has previously been shown that non-thermal (i.e., cold) plasma can successfully decontaminate milk from Escherichia coli. This study investigated the possible biochemical changes to the protein, free fatty acids and volatiles profiles of whole raw milk samples following application of cold plasma. Raw milk was treated with a cold plasma system at intervals of 0, 3, 6, 9, 12, 15 and 20 min. Significant changes were observed for 1 octanol (P < 0.05), 2 heptanone (P < 0.01), 2 hexenal (P < 0.01), 2 octenal (P < 0.05), nonanal and benzaldehyde (P < 0.001). Plasma treatment did not result in significant changes to the lipid composition of raw milk. However, exposure to cold plasma significantly increased the total aldehyde content following 20 min treatment. No significant difference was observed in the total ketone or alcohol levels.     

Variation in fatty acid composition of ewes' milk during continuous transition from dry winter to natural pasture diet

The profile variations of approximately 70 fatty acids (FAs) in milk of ewes consuming total mixed rations (TMR) and grazing natural pasture were determined. Milk of ewes grazing pasture in May and September in the Slovak Republic contained 3-times more conjugated linoleic acid (CLA) (P < 0.001) and trans-vaccenic acid (TVA) (P < 0.001), and 2-times more α-linolenic acid (ALA) (P < 0.001) than that of ewes fed TMR. A doubling of the CLA and TVA contents of milk of ewes fed with TMR was achieved by changing to meadow hay containing a higher ALA content. Less availability of vegetation and lower ALA pasture content in summer (P < 0.001) caused a 2-fold decrease (P < 0.001) of CLA and TVA contents when compared with data obtained in May. With grass re-growth and an increase in pasture ALA content in September, the CLA and TVA contents rose to values similar to those obtained in May (P > 0.05). No significant differences in the FA composition in milk of ewes grazing on summer pasture at higher altitude were observed.     

Chemical composition,protein fraction and fatty acid profile of donkey milk during lactation

The chemical composition, fatty acid and protein fraction of donkey milk samples from domestic (Arcadian) breed were assessed from the 30th day to the 210th day of lactation. Total fat, proteins and minerals (Ca, Mg, K and P) were affected by the stage of lactation. Fatty acids were significantly influenced, with a decrease of 31.4% in saturated fatty acids, an increase of 53.8% in unsaturated fatty acids, a concomitant elevation of polyunsaturated fatty acids and a low n-6/n-3 ratio (2:1). Analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis found, on average, 42.8% casein and 57.2% whey protein. The concentration of lysozyme was between 1.20 and 2.54 mg mL⁻¹, while the highest values were detected by the lysoplate method. Finally, low microbial content and somatic cells were also found in donkey milk with overall average log 4.4 cfu mL⁻¹ and log 4.8 cells mL⁻¹, respectively.     

Effects of somatic cell counts in milk on physical and chemical characteristics of yoghurt

The quality of plain stirred yoghurt produced from whole milk with somatic cell counts (SCC) at low (147,000 cells mL⁻¹), intermediate (434,000 cells mL⁻¹) and high (1,943,000 cells mL⁻¹) levels was examined. Each milk treatment was obtained from selected cows, according to its SCC status and milk composition. Yoghurt samples were analysed on days 1, 10, 20 and 30 after production. Analyses included pH, acidity, fat, lipolysis (expressed as free fatty acids, FFA), proteolysis and apparent viscosity. Viscosity of high SCC yoghurt was higher (P<0.05) than the low SCC yoghurt on days 10, 20 and 30 of storage. High SCC yoghurt also had higher FFA content (P<0.05). SCC did not affect pH, acidity, fat content and proteolysis of the yoghurt (P>0.05). Results indicate that SCC in milk increases the lipolysis in the resulting yoghurt during storage for 30 d.     

Immunomodulatory activity of 5 kDa permeate fractions of casein hydrolysates generated using a range of enzymes in Jurkat T cells and RAW264.7 macrophages

The in vitro bioactivity of 5 kDa ultrafiltration permeate fractions of casein hydrolysates produced using different enzymes were compared. Reverse phase ultra-performance liquid chromatography and gel permeation chromatography showed that the permeates had different physicochemical properties (molecular mass and degree of hydrolysis). The Flavourzyme® permeate had the highest activity in the 2,2’-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay. Cellular antioxidant and immunomodulatory assays showed that none of the permeates exhibited in vitro antioxidant activity, while all permeates significantly (P < 0.05) decreased interleukin-6 (IL-6) production in ConA-stimulated Jurkat T cells at 0.50% (w/v) and LPS-stimulated RAW264.7 cells at 0.05 and 0.50% (w/v). Three permeates, obtained using Flavourzyme®, Flavorpro Whey and trypsin, also significantly (P < 0.05) decreased IL-1β production at 0.05% (w/v) in RAW264.7 cells. Western blot analysis showed that all permeates significantly decreased the expression of the NF-κB subunit, p65, in RAW264.7 cells indicating that anti-inflammatory activity may be associated with this pathway.     

The immunological consequences of pasteurisation: Comparison of the response of human intestinally-derived cells to raw versus pasteurised milk

Microarray technology was used to identify differentially expressed biological signatures in human intestinal cell line (HT-29) exposed to raw versus pasteurised milk; 1041 differentially expressed genes (≥1.3 fold change) were identified (P < 0.001) between exposure groups. These were further identified to be contained within 179 gene ontologies. Genes more highly expressed (599 or 57.5%) in cells exposed to raw milk were predominately contained within immune-based gene ontologies. In contrast, genes showing lower expression in raw milk treated cells (442 or 42.5%) in comparison with pasteurised milk were involved in a broader range of cellular functions. The lowered immune function identified in cells exposed to pasteurised milk was intriguing and suggests that raw milk may be capable of inducing certain aspects of the immune system, including processes involved in T and B cell function/development. The results may indicate an alteration in the immunomodulatory potential of milk following pasteurisation.     

Purification and anti-tumour activity of cyanidin-3-O-glucoside from Chinese bayberry fruit

Chinese bayberry (Myrica rubra Sieb. & Zucc.) is a rich source of cyanidin-3-O-glucoside (C3G). Purification of C3G was established by using high-speed counter-current chromatography (HSCCC) at 520 nm. The optimised flow rate was 2 ml/min and rotational speed was 850 rpm, and n-butanol-methyl tertiary butyl ether-acetonitrile-trifluoroacetic acid-water (30:20:40:1:100, v/v/v/v/v) solvent showed the highest partition coefficient (K = 1.50) to purify C3G. Different HSCCC fractions showed significant different anti-tumour effect on SGC7901, AGS, and BGC823 gastric cells, which were correlated with their C3G concentrations and DPPH^• scavenging activities. Cells treated with C3G resulted in reduced cell proliferation, decreased cell adherence, and abnormal morphological changes characteristics of apoptosis, all in dose-dependent manners. In addition, increased concentrations of C3G treatment resulted in increased inhibition of matrix metalloproteinase 2 (MMP-2) in SGC7901 cells, which may provide important information for the possible mechanism of C3G-induced anti-tumour activity against gastric adenocarcinoma cells.     

Composition, physiochemical properties, nitrogen fraction distribution, and amino acid profile of donkey milk

This study investigated the changes in chemical composition, nitrogen fraction distribution, and AA profile of milk samples obtained during lactation from the Jiangyue breed of donkey in Northwest China. Results showed that donkey milk contained 9.53% total solids, 1.57% protein, 1.16% fat, 6.33% lactose, and 0.4% ash on average, which is more similar to mare and human milk than to the milk of other mammals. Throughout the lactation investigated, pH and density were constant, protein and ash content showed an apparent negative trend (an increase in lactose content during 120 d postpartum, followed by a decrease), fat content exhibited wide variability, and variations in the content and percentage of whey protein, casein, and AA were small. The casein to whey protein ratio of 52:37 was between the lower value of human milk and the higher value of cow milk. Sodium dodecyl sulfate-PAGE results demonstrated that donkey milk is rich in β-lactoglobulin and lysozyme. The percentages of 8 essential AA in protein of donkey milk were 38.2%, higher than those of mare and cow milk; donkey milk also had higher levels of serine (6.2%), glutamic acid (22.8%), arginine (4.6%), and valine (6.5%) and a lower level of cystine (0.4%).     

Cycled high hydrostatic pressure processing of whole and skimmed milk: Effects on physicochemical properties

In this study, we compare the effects of single- and double-cycle HP treatments at 600 MPa on inactivation of the natural microflora and physicochemical properties of whole and skimmed milk of high bacterial load. The results show that two-cycled HP (2 × 2.5 min) was more effective (P < 0.05) on microbial inactivation, and caused similar or slightly less changes (P > 0.05) in physicochemical properties of milk in comparison to single cycled HP (1 × 5 min). In addition to the expected milk protein structure changes, HP at 600 MPa caused only slight effects on milk fat and lactose. Minor decreases in levels of short chain fatty acids were observed with the cycled treatments, and the volatiles in general decreased after HP treatment, depending mostly on the pressure time but also on cycling in skimmed milk. The study confirmed the superior effect of two-cycle HP on microbial inactivation, and shows a slightly better preservation of the physical-chemical milk quality.Industrial relevanceMulti-cycling HP has been shown to be advantageous for microbial inactivation, but limited information is available regarding the effect on milk components in whole milk or skimmed milk. The present study compares the psychochemical properties of whole and skimmed milk processed by multi-cycling in comparison to single cycle HP treatment. Double cycled HP presented a superior effect on microbial inactivation and slightly better preservation of milk quality than one continuous HP.     

The citrus flavonoid nobiletin inhibits proliferation and induces apoptosis in human pancreatic cancer cells in vitro

The cellular effects of nobiletin on human pancreatic cancer cells (PANC-1) and the mechanisms by which nobiletin inhibits the proliferation of these cells were investigated. A MTT assay and flow cytometry were used to examine cell proliferation and apoptosis, respectively. A Western blot assay was used to examine expression levels of the apoptotic proteins bax, bcl-2, and p53. NOB induced apoptosis in these cells via up-regulation of the proapoptotic protein bax and down-regulation of the antiapoptotic proteins bcl-2 and p53. The normal cell cycle of PANC-1 cells was arrested by NOB with a significant increase in the proportion of G0/G1 phase cells (p<0.05) and a significant decrease in the proportion of S phase cells (p<0.05). NOB can inhibit the proliferation of human pancreatic carcinoma cells by inducing apoptosis and arresting the cell cycle progression.     

In vitro gastrointestinal digestion of purified bovine kappa-casein variants A,B, and E: Effects on antioxidant and angiotensin 1-converting enzyme inhibitory capacity

The κ-CN genetic variants A, B, and E were purified from milk from homozygous cows, using isoelectric precipitation followed by calcium precipitation and ion-exchange chromatography. The purified κ-CN variants displayed varying degrees of glycosylation, and were furthermore differentially affected by in vitro gastrointestinal digestion. Both antioxidant and angiotensin 1-converting enzyme inhibitory capacity were significantly increased by digestion, with the antioxidant capacity of the E variant being significantly lower (p < 0.05) than that of the A and B variants. These results suggest that genetic polymophisms in bovine milk protein coding genes may be of significance for nutritional and health-related endpoints.     

Antioxidant and anti-proliferative activity of Rhizoma Smilacis Chinae extracts and main constituents

Rhizoma Smilacis Chinae (RSC) is a widely used herbal material in functional food and folk medicine. In this study, methanol extract (ME), water extract (WE), polysaccharide fraction and ethyl acetate fraction (EF) of RSC were prepared and the constituents were analysed by HPLC. Different antioxidant tests were employed to evaluate the antioxidant activities of RSC extracts and its main constituents, astilbin and chlorogenic acid. The results showed that RSC extracts possessed comparable antioxidant activity to butylated hydroxyanisole in a dose-dependent manner. The radical-scavenging capacity of ME and EF was even stronger than astilbin and chlorogenic acid. The EF and ME of RSC also showed stronger anti-proliferative activity on HepG2 cells than astilbin and chlorogenic acid, with IC₅₀ values of 47 and 32 μg/mL for 24 h treatment, respectively. Flow cytometric analysis revealed that RSC extracts induced cell cycle arrest at G2/M phase and a late apoptosis of the cells.     

The biological activity of fermented milk produced by Lactobacillus casei ATCC 393 during cold storage

Lactobacillus casei ATCC 393 is an important probiotic strain widely known in dairy technology. However, its capability to produce bioactive peptides from milk proteins has not been studied. The viability of the Lb. casei ATCC 393 strain and some physicochemical properties in fermented milk throughout storage for 21 days at 4 °C was evaluated; biological activity, i.e., antioxidant, angiotensin converting enzyme inhibitory and anticancer activities of water soluble extract and its filtrate (< 2 kDa; F1) were determined. Lb. casei counts remained over 9 log cfu g⁻¹ during the storage period in fermented milk. These bioactivities were increased significantly (P < 0.01) during storage. F1 of fermented milk after three weeks of storage showed the highest bioactivity impact. De novo sequencing assay for peptide identification was applied to the mass spectrum of F1. The promising capability of Lb. casei ATCC 393 to release bioactive peptides from milk proteins was demonstrated.     

Evaluation of volatile compounds and free fatty acids in set types yogurts made of ewes’, goats’ milk and their mixture using two different commercial starter cultures during refrigerated storage

Six different types of yogurt were manufactured from Damascus goat milk, Awassi ewe milk and a mixture of equal portions of the 2 species of milk using 2 types of commercial yogurt cultures (CH-1 and YF-3331). Yogurts were chemically analysed at 1, 7, 14 and 21 days of storage. Results showed that cultures significantly affected acetaldehyde (P < 0.05), acetone (P < 0.05) and diacetyl (P < 0.001) contents. Type of milk significantly influenced acetaldehyde (P < 0.05), diacetyl (P < 0.001), acetoin (P < 0.001) and ethanol (P < 0.05) levels. Significant variations occurred in acetaldehyde (P < 0.001) and acetoin (P < 0.05) contents during the storage. Short-chain free fatty acids were the highest in ewes’ milk yogurt made with culture YF-3331, and increased during storage, while the levels of medium-chain free fatty acids, except for decanoic acid, were unchanged and the amount of long-chain free fatty acids decreased during storage. Cultures used and types of milk had no effect on long-chain free fatty acids in yogurts.