Validation of an HPLC Method for Pretreatment of Steviol Glycosides in Fermented Milk.

Steviol glycosides are used in food and beverages worldwide as natural sweeteners, serving as a low-calorie sugar substitute. The acceptable daily intake of steviol is 0-4 mg/kg body weight. The rising demand for dairy products has led to a corresponding increase in the use of steviol glycosides in such products. Therefore, it is important to analyze the levels of steviol glycosides in dairy products. Dairy products have high fat contents and unique emulsion characteristics, conferred by a mixture of fat globules, casein micelles, whey proteins, and numerous other small molecules. These characteristics may interfere with the estimation of steviol glycoside levels; therefore, dairy samples require pretreatment. We aimed to develop an objective test for measuring the levels of steviol glycosides through the development of an efficient pretreatment method. In this study, the steviol glycoside content in dairy products was evaluated by using various methods, and an optimal pretreatment method was determined. We used high-performance liquid chromatography to assess the selectivity, linearity, limit of detection, limit of quantification, accuracy, precision, and recovery rate. Calibration curves were linear in the range of 1-50 mg/kg, with a coefficient of determination of ≥0.999. The limit of detection and limit of quantification were in the ranges of 0.11-0.56 and 0.33-1.69 mg/kg, respectively. The relative standard deviation (%) represents the precision of a measurement. The RSD relative standard deviationof recovery varied between 0.16% and 2.83%, and recovery of the analysis varied between 83.57% and 104.84%. These results demonstrate the reliability of the method for measuring the steviol glycoside content. This method can be used for the simple pretreatment of steviol glycosides and can provide an accurate determination of steviol glycoside content in emulsified food matrices, such as dairy products.

Anti-asthma and antitussive effects of a fermented extract of a mixture of Ramulus mori, Anthriscus sylvestris, and Salvia plebeian.

Respiratory immunity is getting more important recently due to outbreak of respiratory diseases and increasing the concentration of fine dust. The aim of this study was to investigate respiratory protection effect of a fermented extract of medicinal plants (FEMP) containing Ramulus mori, Salvia plebeia, and Anthriscus sylvestris. The expression levels of IL-8 and IL-17 in LPS/poly-L-arginine (PLA) and FEMP-cotreated A549 cells were lower than those in LPS/PLA only-treated cells. The levels of IgE, IL-17, and IL-4 in the bronchoalveolar lavage fluid (BALF) and serum of FEMP-treated mice with ovalbumin/LPS-induced asthma were lower than the control levels. The lung inflammation score and the number of inflammatory cells in the BALF decreased by FEMP treatment. In the citric acid-induced coughing guinea pig, the FEMP treatment decreased the number of coughs. Therefore, FEMP shows anti-asthmatic and antitussive activities without hepatotoxicity and can be used as a compound aiming to improve respiratory health. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00955-3.

Determination of L-Carnitine in Infant Powdered Milk Samples after Derivatization.

Herein, a novel analytical method using a high-performance liquid chromatography-fluorescence detector (HPLC/FLD) is developed for rapidly measuring an L-carnitine ester derivative in infant powdered milk. In this study, solid-phase extraction cartridges filled with derivatized methanol and distilled water were used to effectively separate L-carnitine. Protein precipitation pretreatment was carried out to remove the protein and recover the analyte extract with a high recovery (97.16%-106.56%), following which carnitine in the formula was derivatized to its ester form. Precolumn derivation with 1-aminoanthracene (1AA) was carried out in a phosphate buffer using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) as the catalyst. Method validation was performed following the AOAC guidelines. The calibration curves were linear in the L-carnitine concentration range of 0.1-2.5 mg/L. The lower limit of quantitation and limit of detection of L-carnitine were 0.076 and 0.024 mg/L, respectively. The intra- and interday precision and recovery results were within the allowable limits. The results showed that our method helped reduce the sample preparation time. It also afforded higher resolution and better reproducibility than those obtained by traditional methods. Our method is suitable for detecting the quantity of L-carnitine in infant powdered milk containing a large amount of protein or starch.

Pretreatment methods for analyzing steviol glycosides in diverse food samples.

Steviol glycosides are well-known food sweeteners; their consumption has steadily increased over time. A pretreatment method was developed and validated to better separate rebaudioside A and stevioside from various protein-rich and fatty foods for quantification. This method was applied to soy sauce in liquid type and fish cake and coffee in solid type. Parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision were calculated. Calibration curves were linear in the working range of 5-100 mg/l, with coefficients of determination ≥0.99. The LOD and LOQ were in the ranges of 0.16-0.39 and 0.52-1.28 mg/kg, respectively. The percentage recoveries of the fortified samples were in the 88.01%-103.09% range, and the relative standard deviation was <10%. Method validation predicted a desirable accuracy, linearity, and precision. Therefore, the developed method can be practically applied for the quantitation of steviol glycosides in various foods, including soy sauce in liquid type and fish cake and coffee in solid type.

Development of Pretreatment Method for Analysis of Vitamin B12 in Cereal Infant Formula using Immunoaffinity Chromatography and High-Performance Liquid Chromatography.

Vitamin B12 deficiency may lead to serious health issues in both infants and adults. A simple analytical method involving sample pretreatment with enzyme, followed by cyanide addition under acidic conditions; separation on an immunoaffinity column; and high-performance liquid chromatography (HPLC) was developed for the rapid detection and quantitation of vitamin B12 in powdered milk. Detection limit and powdered milk recovery were determined by quantitative analysis. The limits of detection and quantitation were 2.71 and 8.21 µg/L, respectively. Relative standard deviations of the intra-day and inter-day precisions varied in the ranges of 0.98%-5.31% and 2.16%-3.90%, respectively. Recovery of the analysis varied in the range of 83.41%-106.57%, suggesting that the values were acceptable. Additionally, vitamin B12 content and recovery in SRM 1849a were 54.10 µg/kg and 112.24%, respectively. Our results suggested that the analytical method, including the sample pretreatment step, was valid. This analytical method can be implemented in many laboratory-scale experiments that seek to save time and labor. Therefore, this study shows that immunoaffinity-HPLC/ultraviolet is an acceptable technique for constructing a reliable database on vitamin B12 in powdered milk containing starch as well as protein and/or fat in high amounts.

Determining the Reuse of Frying Oil for Fried Sweet and Sour Pork according to Type of Oil and Frying Time.

Food Codex regulations have set freshness limits for oils used to fry food, such as potato and fish products, and fried food itself; however, no such freshness limits have been set for meat products, such as sweet and sour pork. The freshness standard suggest that acid values (AVs) and peroxide values (POVs) for frying oil should be less than 2.5 and 50, respectively, whereas AVs and POVs for common fried food should be less than 5.0 and 60, respectively. Therefore, in this study, we investigate the effect of the number of frying cycles on oxidation-promoted changes in the oils used to fry sweet and sour pork and fried food itself during repeated frying over 10 d by determining their AVs and POVs, which were found to be highly correlated. Soybean, canola, palm, and pork lard oils could be reused approximately 37, 32, 58, and 87 times, respectively, to fry sweet and sour pork based on oil freshness, and 78, 78, 81, and 286 times, respectively, based on the freshness of fried food. Our data may help establish food-quality regulations for oils used to fry animal-based foods.

Transfer rates of pathogenic bacteria during pork processing.

We examined the rates of pathogenic bacterial cross-contamination from gloves to meat and from meat to gloves during pork processing under meat-handling scenarios in transfer rate experiments of inoculated pathogens. The inoculated pork contained ~5-6 Log10 CFU/g pathogenic bacteria like Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes), and Salmonella enterica subsp. enterica (Sal. enteritidis). On cotton gloves, after cutting the pork, the cutting board, knife, and cotton gloves showed 3.07-3.50, 3.29-3.92 and 4.48-4.86 Log10 CFU/g bacteria. However, when using polyethylene gloves, fewer bacteria (3.12-3.75, 3.20-3.33, and 3.07-3.97 Log10 CFU/g, respectively) were transferred. When four pathogens (6 Log10 CFU/g) were inoculated onto the gloves, polyethylene gloves showed a lower transition rate (cutting board 2.47-3.40, knife 2.01-3.98, and polyethylene glove 2.40-2.98 Log10 CFU/g) than cotton gloves. For cotton gloves, these values were 3.46-3.96, 3.37-4.06, and 3.55-4.00 Log10 CFU/g, respectively. Use of cotton gloves, polyethylene gloves, knives and cutting boards for up to 10 hours in a meat butchering environment has not exceeded HACCP regulations. However, after 10 h of use, 3.09, 3.27, and 2.94 Log10 CFU/g of plate count bacteria were detected on the cotton gloves, cutting board, and knives but polyethylene gloves showed no bacterial count. Our results reveal the transfer efficiency of pathogenic bacteria and that gloved hands may act as a transfer route of pathogenic bacteria between meat and hands. The best hand hygiene was achieved when wearing polyethylene gloves. Thus, use of polyethylene rather than cotton gloves reduces cross-contamination during meat processing.

Determination of Shelf Life Model of Pork Cutlet and Pork Lard during Accelerated Storage Conditions.

This study was carried out to establish shelf life for pork cutlet of ground meat and pork lard by using various quality indicators and to understand how quality changes in these products are accelerated by temperature. The samples were selected and purchased from markets in Korea, and the chosen quality indicators were total aerobic counts and coliform group in microbiological analyses, thiobarbituric acid reactive substances assay, volatile basic nitrogen, pH, acid value, and peroxide value in physical chemical analyses, and sensory evaluation. The pork cutlet samples were stored at -18℃, -6℃, and -1℃, whereas pork lard samples were stored at 10℃, 25℃, 35℃, and 45℃. These temperature conditions were set to real distribution conditions. The samples were then analyzed using various models including of reaction orders, arrhenius equation, and Q10 value. The quality limits for each sample were calculated, and shelf life was estimated. The results of this experiment highlighted the importance of temperature control during the distribution process of these products and revealed that temperature is a useful parameter for the establishment of a basic database for shelf life.

Probiotic Potential of Enterococcus faecium Isolated from Chicken Cecum with Immunomodulating Activity and Promoting Longevity in Caenorhabditis elegans.

Probiotics, including Enterococcus faecium, confer a health benefit on the host. An Enterococcus strain was isolated from healthy chicken cecum, identified as E. faecium by 16S rDNA gene sequence analysis, and designated as E. faecium L11. To evaluate the potential of E. faecium L11 as a probiotic, the gastrointestinal tolerance, immunomodulatory activity, and lifespan extension properties of the strain were assayed. E. faecium L11 showed >66% and >62% survival in artificial gastric juice (0.3% pepsin, pH 2.5) and simulated small intestinal juice (0.5% bile salt and 0.1% pancreatin), respectively. Heat-killed E. faecium L11 significantly (p < 0.05) increased immune cell proliferation compared with controls, and stimulated the production of cytokines (IL-6 and TNF-α) by activated macrophages obtained from ICR mice. In addition, E. faecium L11 showed a protective effect against Salmonella Typhimurium infection in Caenorhabditis elegans. In addition, feeding E. faecium L11 significantly (p < 0.05) extended the lifespan of C. elegans compared with the control. Furthermore, genes related to aging and host defense were upregulated in E. faecium L11-fed worms. In conclusion, E. faecium L11, which prolongs the lifespan of C. elegans, may be a potent probiotic supplement for livestock.

Lifespan Extension of Caenorhabditis elegans by Butyricicoccus pullicaecorum and Megasphaera elsdenii with Probiotic Potential.

Butyricicoccus pullicaecorum and Megasphaera elsdenii inhabit the human intestine and have probiotic potential. The aim of this study was to evaluate the effects of B. pullicaecorum and M. elsdenii on the lifespan of Caenorhabditis elegans. They significantly (P < 0.05) extended the lifespan of C. elegans compared with Escherichia coli OP50, a standard food for the worm. Analysis of age-related biomarkers such as lipofuscin, body size, and locomotory activity showed that they retarded aging. They all failed to extend the lifespan of daf-12 or dbl-1 loss-of-function C. elegans mutants compared with E. coli OP50-fed worms. However, the increase in lifespan was observed in daf-16, jnk-1, pmk-1, and skn-1 mutants. Moreover, they increased the resistance of C. elegans to a human pathogen, Salmonella typhimurium. In conclusion, B. pullicaecorum and M. elsdenii extend the lifespan of C. elegans via the transforming growth factor-beta (TGF-ß) pathway associated with anti-inflammatory processes in the innate immune system.

Predicting Shelf-life of Ice Cream by Accelerated Conditions.

Shelf-life is defined as the amount of time during which a food product retains its desired sensory, chemical, and physical characteristics while remaining safe for consumption. The food industry needs to rapidly obtain the necessary information for determining the shelf life of its products. Here we studied the approaches available for conducting accelerated shelf-life tests. Accelerated shelf-life testing is applied to a variety of products to rapidly estimate change in characteristics with time. The aim of this work was to use accelerated shelf-life testing to study the changes in pH, microbiology, and sensory characteristics of ice cream by the application of a kinetic approach and, based on the observations, to estimate its shelf life. As per the current law, there is no shelf life on ice cream. Our results suggest that the shelf life of an ice cream sample was 24.27 months at -18℃, 2.29 months at -6℃, 0.39 months at -1℃, and 0.15 months at 4℃. Results of this study suggest that a set expiration date on ice cream might also contribute to effective management of ice cream characteristics in the retail chilled chain.

Cherry tomato supplementation increases the area of the intestinal mucosa and the number of muscle layers in rats.

Tomatoes act as prebiotics in the gut. The effects of cherry tomatoes on gastrointestinal health have not yet been studied. Four cherry tomato supplementation diets (CTSDs) were prepared from the juice and cake of fresh and processed (heat-treated) cherry tomatoes. The contents of the gut and histological changes in the cecum and intestine were analyzed at 4weeks in rats fed CTSDs. The lactic acid bacteria level in fecal contents of rats fed CTSDs increased compared with the control. The gut length was longer in rats fed CTSDs than that in control animals. In addition, the cecal propionate level significantly increased (p<0.05), and acetate and butyrate levels decreased compared with control animals, however, regardless of the type of CTSD, the total concentration of short chain fatty acids (SCFAs) in all rats fed different CTSDs was similar with the control. The thicknesses of the mucosa and muscle of the cecum and colon increased in rats fed CTSDs compared with the control. CTSDs increased the area of the mucosa and the number of muscle layers in the intestine and cecum of rats, which strengthened the barrier function and promoted gastrointestinal health.

Effect of water-soluble fraction of cherry tomatoes on the adhesion of probiotics and Salmonella to intestinal epithelial cells.

BACKGROUND: Tomato is one of the most consumed vegetables in the world and contains many valuable nutritional components. Here we investigate the prebiotic effects of cherry tomatoes for improving gut health. RESULTS: Water-soluble dietary fiber was prepared from fresh and processed (heat treatment at 80 °C for 15 min) cherry tomato samples, each with and without Viscozyme L treatment. In the adhesion assays, all water-soluble dietary fiber samples improved adhesion of probiotics (Lactobacillus rhamnosus and Bifidobacterium bifidum) to intestinal epithelial cells (Caco-2 cells). Heat treatment in the preparation of juice from cherry tomatoes showed no significant effect on the adhesion of probiotics to Caco-2 cells. The oligofructose content of samples affected the intestinal adhesion of probiotic bacteria, with higher oligosaccharide concentrations associated with greater adhesion of probiotics and more inhibition of the adhesion of pathogens to Caco-2 cells. CONCLUSION: The present results suggest that cherry tomato can act as a prebiotic, with oligofructose potentially being one of its major prebiotic components.

Chemical characterization of tomato juice fermented with bifidobacteria.

The objective of this research was to characterize the chemical properties of tomato juice fermented with bifidobacterial species. Tomato juice was prepared from fresh tomatoes and heated at 100 degrees C prior to fermentation. Bifidobacterium breve, Bifidobacterium longum, and Bifidobacterium infantis were inoculated in tomato juice and kept at 35 to 37 degrees C for up to 6 h. Fructooligosaccharide (FOS) was added to tomato juice prior to fermentation. The analyses for brix, total titratable acidity (TTA), pH, color, and lycopene content were conducted to characterize tomato juices fermented with bifidobacterial species. Heat treatment of tomato juice did not cause any significant changes in brix, pH, and TTA. Only the redness of tomato juice was significantly increased, as the heating time increased to 30 min. The tomato juices fermented with B. breve and B. longum exhibited significant decreases in pH (3.51 and 3.80, respectively) and significant increases in TTA (13.50 and 12.50, respectively) (P < 0.05). B. infantis did not cause any significant change in the chemical properties of tomato juice. The addition of FOS further improved the fermentation of tomato juice by bifidobacterial species. The lycopene contents of tomato juice were significantly increased from 88 to 113 microg/g by heat treatment at 100 degrees C (P < 0.05), however did not exhibit any significant change after fermentation with bifidobacterial species.

Hot-water extract from mycelia of Cordyceps sinensis as a substitute for antibiotic growth promoters.

Broiler chicks were orally dosed with a hot-water extract of mycelia from Cordyceps sinensis (CS-HW) to assess possible substitution of Avilamycin as an antibiotic growth promoter (AGP). The growth performance (body weight gain and survivability) and the health index (the microflora in the small intestines and the antibody titer to Newcastle disease virus) of chicks were significantly improved in the CS-HW (600 mg/kg diet) and the Avilamycin (20 mg/kg diet) fed group in comparison with the control group (p < 0.05). The Avilamycin-fed group and the CS-HW-fed group had similar growth performances but the latter gave a better microbial flora in the small intestines. These results indicate that CS-HW enhances the physiological activity in chicks and can be used as a substitute for AGPs.

Antifatigue and antistress effect of the hot-water fraction from mycelia of Cordyceps sinensis.

This study was conducted to investigate the chemical component of the hot water (HW) fraction of mycelia of Cordyceps sinensis and its antifatigue and antistress effect against a stimulus in vivo using rats and mice. The growth of mycelia reached a maximum level of 31.6 g/l after 120 h of incubation. The main chemical composition of the HW fraction of mycelia of C. sinensis was found to be carbohydrate (78.9%) with 5% moisture. The swimming endurance capacity of mice orally administered with the HW fraction (150 and 300 mg/kg/d, respectively) was significantly prolonged from 75 to 90 min with a lessening of fatigue. When the HW fraction (150 mg/kg/d) was given to rats for 8 d including a 48 h stress period, the weight changes of the adrenal gland, spleen, thymus, and thyroid, which is an index of stress, were suppressed. The HW fraction also significantly inhibited the increase in total cholesterol and the decrease in alkaline phosphatase levels as biochemical parameters of immobilization stress in rats.

Hypocholesterolemic effect of hot-water extract from mycelia of Cordyceps sinensis.

This study was conducted to investigate the hypocholesterolemic effect of the hot-water fraction (HW) from cultured mycelia of Cordyceps sinensis in a 5 l fermenter. The composition of HW was mainly carbohydrate (83.9%) and protein (11.8%) on a dry basis, and the carbohydrate of HW consisted of glucose, mannose, galactose, and arabinose in the molecular ratio of 1.0 : 0.8 : 0.5 : 0.1, respectively. In mice fed a cholesterol-free diet and those fed a cholesterol-enriched diet, body and liver weights were not significantly different from those of the controls. The serum total cholesterol (TC) of all mice groups administered HW (150 and 300 mg/kg/d, respectively) with the cholesterol-enriched diet decreased more than in the control group. Among the mice fed the cholesterol-enriched diet, HW also increased the high-density lipoprotein (HDL) cholesterol level, but decreased the very low-density lipoprotein plus low-density lipoprotein (VLDL+LDL) cholesterol level. The changes in HDL- and VLDL+LDL-cholesterol levels consequently decreased the atherogenic value. The results indicate that HW in rats administered a cholesterol-enriched diet decreased the plasma cholesterol level. The 300 mg/kg dose had a significant effect on the serum TC level.

Activation of macrophages and the intestinal immune system by an orally administered decoction from cultured mycelia of Cordyceps sinensis.

The effects of an orally administered hot-water extract (HW) from cultured mycelia of Cordyceps sinensis on the activation of macrophages and the intestinal immune system were studied in mice. The general composition of HW was 83.9% carbohydrate, 11.8% protein, 1.9% lipid and 2.4% ash, and the carbohydrates were mainly composed of glucose, mannose, galactose and arabinose (molar ratio of 1.0:0.8:0.5:0.1). HW stimulated the activation (1.7-fold of the saline control) of macrophages and IL-6 production (1.5-fold) at 2.0 g/kg/day. Analyzing the culture supernatant of Peyer's patch cells from C3H/HeJ mice that had been fed with HW at 1.0 g/kg/day for 7 days indicated that the bone marrow cells had significantly proliferated (1.9-fold). In addition, the amounts of GM-CSF and IL-6 in the culture supernatant of Peyer's patch cells at the same dose were significantly increased (1.8-fold and 2.2-fold, respectively). These results indicate that an oral administration of HW may modulate IL-6 production by the activation of macrophages, and also enhance the secretion of hematopoietic growth factors such as GM-CSF and IL-6 from Peyer's patch cells. Since such cytokines as GM-CSF and IL-6 from Peyer's patch cells act on the systemic immune system, it can be assumed that orally administered HW modulated not only the local but also systemic immune system.

Effects of SO2 and NO on growth of Chlorella sp. KR-1.

Effects of the toxic compounds in flue gas, SOx and NOx, on growth of Chlorella sp. KR-1 have been determined. Although growth of KR-1 was suppressed by the toxic compounds, KR-1 exhibited excellent tolerances to SOx compared to other algal strains. When Chlorella KR-1 was cultured with the model gas containing 60 ppm SO2, the linear growth rate was 1.24 g/l day which is about 25% lower than that of the control culture aerated with the gas mixture containing no toxic compounds, SO2 and NO. KR-1 could grow even with the model gas containing 100 ppm SO2 and the linear growth rate of KR-1 in the culture was 0.78 g/l day. The period for lag phase was increased with increasing of SO2 concentration that also resulted in the decrease of the linear growth rate and the maximum cell concentration. Direct CO2 fixation by Chlorella KR-1 has been successfully done using actual flue gases from a liquified natural gas (LNG)- or diesel-fueled boiler. These results indicated that Chlorella KR-1 may be applied for direct CO2 fixation from actual flue gas.

Host immune responses against hog cholera virus in pigs treated with an ionized alkali mineral complex.

To determine the immune responses in pigs to hog cholera virus after treatment with an ionized alkali mineral complex (IAMC), 40 healthy pigs (28-32 days old) from a commercial swine farm were purchased and housed into 4 groups (n=10 each). All pigs were vaccinated intramuscularly (1 ml) with an attenuated live hog cholera virus (HCV, LOM strain) at 28-32 days old and challenged with a virulent hog cholera virus at 8 weeks after vaccination. Each group was treated with PowerFeel sprayed diet as 0.05% (w/w) in a final concentration (T-1, n=10), a diet mixed with SuperFeed as 3% (w/w) in a final concentration (T-2, n=10), or a diluted PowerFeel solution (1:500, v/v) as drinking water (T-3, n=10), respectively. A group (n=10) served as a non-treated control. Proportions of expressing CD2+ and CD8+ cells increased significantly (p<, 0.05) at 8-week post-application. Mean antibody titers of each group against HCV gradually increased to higher levels after vaccination and with challenge of the virulent virus. In conclusion, the IAMC-treated diets can be helpful for the improvement of growth in pigs with proper vaccination program, while the IAMC-treated diets have no effects on the clinical protection against hog cholera.