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1.  The Antimetastatic and Antiangiogenesis Effects of Kefir Water on Murine Breast Cancer Cells 
Integrative Cancer Therapies  2016;15(4):NP53-NP66.
Background. Kefir is a unique cultured product that contains beneficial probiotics. Kefir culture from other parts of the world exhibits numerous beneficial qualities such as anti-inflammatory, immunomodulation, and anticancer effects. Nevertheless, kefir cultures from different parts of the world exert different effects because of variation in culture conditions and media. Breast cancer is the leading cancer in women, and metastasis is the major cause of death associated with breast cancer. The antimetastatic and antiangiogenic effects of kefir water made from kefir grains cultured in Malaysia were studied in 4T1 breast cancer cells. Methods. 4T1 cancer cells were treated with kefir water in vitro to assess its antimigration and anti-invasion effects. BALB/c mice were injected with 4T1 cancer cells and treated orally with kefir water for 28 days. Results. Kefir water was cytotoxic toward 4T1 cells at IC50 (half-maximal inhibitory concentration) of 12.5 and 8.33 mg/mL for 48 and 72 hours, respectively. A significant reduction in tumor size and weight (0.9132 ± 0.219 g) and a substantial increase in helper T cells (5-fold) and cytotoxic T cells (7-fold) were observed in the kefir water–treated group. Proinflammatory and proangiogenic markers were significantly reduced in the kefir water–treated group. Conclusions. Kefir water inhibited tumor proliferation in vitro and in vivo mainly through cancer cell apoptosis, immunomodulation by stimulating T helper cells and cytotoxic T cells, and anti-inflammatory, antimetastatic, and antiangiogenesis effects. This study brought out the potential of the probiotic beverage kefir water in cancer treatment.
doi:10.1177/1534735416642862
PMCID: PMC5739168  PMID: 27230756
kefir; kefir water; breast cancer; 4T1 cell; antimetastasis; antiangiogenesis; immunomodulatory
2.  Optimization of processing conditions to improve antioxidant activities of apple juice and whey based novel beverage fermented by kefir grains 
A central composite design (CCD) was used to evaluate the effects of fermentation temperature (20–30 ºC) and kefir grains amount (2–8%w/v) on total phenolic content and antioxidant activities of apple juice and whey based novel beverage fermented by kefir grains. The response surface methodology (RSM) showed that the significant second-order polynomial regression equation with high R2 (>0.86) was successfully fitted for all response as function of independent variable. The overall optimum region was found to be at the combined level of 7.56%w/v kefir grains and temperature of 24.82 ºC with the highest value for total phenolic content (TPC) and antioxidant activities. At this optimum point TPC, 1, 1-Diphenyl-2-picrylhydrazyl radical scavenging, metal chelating effect, reducing power, inhibition of linoleic acid autoxidation and inhibition of ascorbate autoxidation were 165.02 mgGA/l, 0.38 ml/1 ml, 0.757 (absorbance at 700 nm), 46.12 %, 65.33 % and 21 %, respectively. No significant difference (p < 0.05) was found between actual values and predicated values.
doi:10.1007/s13197-014-1397-4
PMCID: PMC4444860  PMID: 26028723
Apple juice; Whey; Kefir; Fermentation; Total phenolic content; Antioxidant activities
3.  Functional Resilience and Response to a Dietary Additive (Kefir) in Models of Foregut and Hindgut Microbial Fermentation In Vitro 
Stability in gut ecosystems is an important area of study that impacts on the use of additives and is related with several pathologies. Kefir is a fermented milk drink made with a consortium of yeast and bacteria as a fermentation starter, of which the use as additive in companion and livestock animals has increased in the last few years. To investigate the effect of kefir milk on foregut and hindgut digestive systems, an in vitro approach was followed. Either rumen fluid or horse fecal contents were used as a microbial inoculate and the inclusion of kefir (fresh, autoclaved, or pasteurized) was tested. Gas production over 72 h of incubation was recorded and pH, volatile fatty acids (VFAs), lactate and ammonia concentration as well as lactic acid (LAB) and acetic acid bacteria, and yeast total numbers were also measured. Both direct and indirect (by subtracting their respective blanks) effects were analyzed and a multivariate analysis was performed to compare foregut and hindgut fermentation models. Addition of kefir boosted the fermentation by increasing molar concentration of VFAs and ammonia and shifting the Acetate to Propionate ratio in both models but heat processing techniques like pasteurization or autoclaving influenced the way the kefir is fermented and reacts with the present microbiota. In terms of comparison between both models, the foregut model seems to be less affected by the inclusion of Kefir than the hindgut model. In terms of variability in the response, the hindgut model appeared to be more variable than the foregut model in the way that it reacted indirectly to the addition of different types of kefir.
doi:10.3389/fmicb.2017.01194
PMCID: PMC5487516  PMID: 28702019
microbial stability; digestive system; gut fermentation; kefir; in vitro
4.  Microbiological, technological and therapeutic properties of kefir: a natural probiotic beverage 
Brazilian Journal of Microbiology  2013;44(2):341-349.
Kefir is a fermented milk beverage produced by the action of bacteria and yeasts that exist in symbiotic association in kefir grains. The artisanal production of the kefir is based on the tradition of the peoples of Caucasus, which has spread to other parts of the world, from the late 19th century, and nowadays integrates its nutritional and therapeutic indications to the everyday food choices of several populations. The large number of microorganisms present in kefir and their microbial interactions, the possible bioactive compounds resulting of microbial metabolism, and the benefits associated with the use this beverage confers kefir the status of a natural probiotic, designated as the 21th century yoghurt. Several studies have shown that kefir and its constituents have antimicrobial, antitumor, anticarcinogenic and immunomodulatory activity and also improve lactose digestion, among others. This review includes data on the technological aspects, the main beneficial effects on human health of kefir and its microbiological composition. Generally, kefir grains contain a relatively stable and specific microbiota enclosed in a matrix of polysaccharides and proteins. Microbial interactions in kefir are complex due to the composition of kefir grains, which seems to differ among different studies, although some predominant Lactobacillus species are always present. Besides, the specific populations of individual grains seem to contribute to the particular sensory characteristics present in fermented beverages. This review also includes new electron microscopy data on the distribution of microorganisms within different Brazilian kefir grains, which showed a relative change in its distribution according to grain origin.
doi:10.1590/S1517-83822013000200001
PMCID: PMC3833126  PMID: 24294220
probiotic; microbial diversity; kefir grains; fermented milk; kefir
5.  Three measures of physical rehabilitation effectiveness in elderly patients: a prospective, longitudinal, comparative analysis 
BMC Geriatrics  2015;15:142.
Background
Rehabilitation success is measured by instruments that assess performance of activities of daily living. Guidelines on the use and choice of these instruments are lacking. The present study aimed to analyse prognostic indicators of physical rehabilitation effectiveness in elderly patients according to three rehabilitation impact indices.
Methods
Prospective, longitudinal study in a post-acute care unit. The study included rehabilitation-eligible deconditioned elderly in-patients prospectively admitted to post-acute care (n = 685, aged 83.2 ± 8.3 years, mean length of stay 15 ± 9.2 days).
Data Collection: Premorbid health status variables (PHSV): age, sex, comorbidity (Charlson index), medical history (heart failure, pulmonary disease, cerebrovascular disease, dementia), previous living situation and pre-admission functional status (premorbid Lawton and Barthel indices). Admission health status variables (AHSV): main diagnoses, referral source, physical (Barthel-adm) and cognitive function (Pfeiffer test), undernutrition and dysphagia.
Outcome Measures: Absolute functional gain (AFG, admission-to-discharge Barthel change), relative functional gain (RFG, achieved percentage of potential gain) and rehabilitation efficiency index (REI, AFG over length of stay). Univariate analysis considered these parameters, along with PHSV and AHSV. Multivariate logistic regression analysis was performed for AFG ≥20, RFG ≥35 % and REI ≥ 0.50.
Results
Greater AFG was associated with 14 variables, 8 PHSV (57.1 %) and 6 AHSV (42.8 %); greater RFG with 9 variables, 3 PHSV (33.3 %) and 6 AHSV (66.6 %); and REI with 9 variables, 4 PHSV (44.4 %) and 5 AHSV (55.5 %). Mean AFG value was 34.5 ± 15.8 in patients who achieved complete recovery (RFG 100 %, n = 189, 27.5 %) and 35.3 ± 15.0 (p = 0.593) in the remaining patients (n = 311, 45.4 %). In multivariate analysis, only Barthel-adm was related to all three rehabilitation impact indices.
Conclusions
Both premorbid and acute-process variables have a greater impact on AFG and REI, compared to RFG. Although AFG gives information about the degree of reduction in dependence, it does not provide clinical information about post-rehabilitation functional status (mean AFG values did not differ between patients with and without complete recovery). A future implication for evaluating rehabilitation effectiveness in elderly patients is to recommend RFG corrected by premorbid Barthel score, which is less affected by previous health conditions, as the optimum method to assess the degree to which maximum potential improvement was achieved.
Electronic supplementary material
The online version of this article (doi:10.1186/s12877-015-0138-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12877-015-0138-5
PMCID: PMC4627405  PMID: 26515028
Geriatric rehabilitation; Functional recovery; Elderly; Rehabilitation impact index; Post-acute
6.  Microbial Species Diversity, Community Dynamics, and Metabolite Kinetics of Water Kefir Fermentation 
Water kefir is a sour, alcoholic, and fruity fermented beverage of which the fermentation is started with water kefir grains. These water kefir grains consist of polysaccharide and contain the microorganisms responsible for the water kefir fermentation. In this work, a water kefir fermentation process was followed as a function of time during 192 h to unravel the community dynamics, the species diversity, and the kinetics of substrate consumption and metabolite production. The majority of the water kefir ecosystem was found to be present on the water kefir grains. The most important microbial species present were Lactobacillus casei/paracasei, Lactobacillus harbinensis, Lactobacillus hilgardii, Bifidobacterium psychraerophilum/crudilactis, Saccharomyces cerevisiae, and Dekkera bruxellensis. The microbial species diversities in the water kefir liquor and on the water kefir grains were similar and remained stable during the whole fermentation process. The major substrate, sucrose, was completely converted after 24 h of fermentation, which coincided with the production of the major part of the water kefir grain polysaccharide. The main metabolites of the fermentation were ethanol and lactic acid. Glycerol, acetic acid, and mannitol were produced in low concentrations. The major part of these metabolites was produced during the first 72 h of fermentation, during which the pH decreased from 4.26 to 3.45. The most prevalent volatile aroma compounds were ethyl acetate, isoamyl acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate, which might be of significance with respect to the aroma of the end product.
doi:10.1128/AEM.03978-13
PMCID: PMC3993195  PMID: 24532061
7.  Combination Therapy in Treatment of Experimental Pulmonary Aspergillosis: In Vitro and In Vivo Correlations of the Concentration- and Dose- Dependent Interactions between Anidulafungin and Voriconazole by Bliss Independence Drug Interaction Analysis▿  
We studied the antifungal activity of anidulafungin (AFG) in combination with voriconazole (VRC) against experimental invasive pulmonary aspergillosis (IPA) in persistently neutropenic rabbits and further explored the in vitro and in vivo correlations by using Bliss independence drug interaction analysis. Treatment groups consisted of those receiving AFG at 5 (AFG5 group) and 10 (AFG10 group) mg/kg of body weight/day, VRC at 10 mg/kg every 8 h (VRC group), AFG5 plus VRC (AFG5+VRC group), and AFG10 plus VRC (AFG10+VRC group) and untreated controls. Survival throughout the study was 60% for the AFG5+VRC group, 50% for the VRC group, 27% for the AFG10+VRC group, 22% for the AFG5 group, 18% for the AFG10 group, and 0% for control rabbits (P < 0.001). There was a significant reduction of organism-mediated pulmonary injury, measured by infarct scores, lung weights, residual fungal burdens, and galactomannan indexes, in AFG5+VRC-treated rabbits versus those treated with AFG5 and VRC alone (P < 0.05). In comparison, AFG10+VRC significantly lowered only infarct scores and lung weights in comparison to those of AFG10-treated animals (P < 0.05). AFG10+VRC showed no significant difference in other outcome variables. Significant Bliss synergy was found in vivo between AFG5 and VRC, with observed effects being 24 to 30% higher than expected levels if the drugs were acting independently. These synergistic interactions were also found between AFG and VRC in vitro. However, for AFG10+VRC, only independence and antagonism were observed among the outcome variables. We concluded that the combination of AFG with VRC in treatment of experimental IPA in persistently neutropenic rabbits was independent to synergistic at a dosage of 5 mg/kg/day but independent to antagonistic at 10 mg/kg/day, as assessed by Bliss independence analysis, suggesting that higher dosages of an echinocandin may be deleterious to the combination.
doi:10.1128/AAC.00329-09
PMCID: PMC2687223  PMID: 19307368
8.  Kefir induces cell-cycle arrest and apoptosis in HTLV-1-negative malignant T-lymphocytes 
Background:
Adult lymphoblastic leukemia (ALL) is a malignancy that occurs in white blood cells. The overall cure rate in children is 85%, whereas it is only 40% in adults. Kefir is an important probiotic that contains many bioactive ingredients, which give it unique health benefits. It has been shown to control several cellular types of cancer.
Purpose:
The present study investigates the effect of a cell-free fraction of kefir on CEM and Jurkat cells, which are human T-lymphotropic virus type I (HTLV-1)-negative malignant T-lymphocytes.
Methods:
Cells were incubated with different kefir concentrations. The cytotoxicity of the compound was evaluated by determining the percentage viability of cells. The effect of all the noncytotoxic concentrations of kefir on the proliferation of CEM and Jurkat cells was then assessed. The levels of transforming growth factor-alpha (TGF-α), transforming growth factor- beta1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), and MMP-9 mRNA upon kefir treatment were then analyzed using reverse transcriptase polymerase chain reaction (RT-PCR). Finally, the growth inhibitory effects of kefir on cell-cycle progression/apoptosis were assessed by Cell Death Detection (ELISA) and flow cytometry.
Results:
The maximum cytotoxicity recorded after 48-hours treatment with 80 μg/μL kefir was only 42% and 39% in CEM and Jurkat cells, respectively. The percent reduction in proliferation was very significant, and was dose-, and time-dependent. In both cell lines, kefir exhibited its antiproliferative effect by downregulating TGF-α and upregulating TGF-β1 mRNA expression. Upon kefir treatment, a marked increase in cell-cycle distribution was noted in the preG1 phase of CEM and Jurkat cells, indicating the proapoptotic effect of kefir, which was further confirmed by Cell Death Detection ELISA. However, kefir did not affect the mRNA expression of metalloproteinases needed for the invasion of leukemic cell lines.
Conclusion:
In conclusion, kefir is effective in inhibiting proliferation and inducing apoptosis of HTLV-1-negative malignant T-lymphocytes. Therefore, further in vivo investigation is highly recommended.
doi:10.2147/CMR.S15109
PMCID: PMC3064404  PMID: 21448298
apoptosis; cancer; CEM; Jurkat; kefir; leukemia
9.  Fermentation process for production of apple-based kefir vinegar: microbiological, chemical and sensory analysis 
Brazilian Journal of Microbiology  2017;48(3):592-601.
The aim of this study was to develop a kefir apple-based vinegar and evaluate this fermentation process using new methodology with Biospeckle Laser. Brazilian kefir grains were inoculated in apple must for vinegar production. In this study, the microbial community present in kefir, and correspondent vinegar, was investigated using Matrix Assisted Laser Desorption/Ionization – Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. Saccharomyces cerevisiae, Lactobacillus paracasei, Lactobacillus plantarum, Acetobacter pasteurianus and Acetobacter syzygii were the microbial species identified. S. cerevisiae, L. plantarum, A. pasteurianus and A. syzygii were found in smaller quantities at the beginning of the alcoholic fermentation, but were found throughout the alcoholic and acetic fermentation. Kefir grains were able to utilize apple must as substrate to produce ethanol, and acetic acid. Acetate, volatile alcohols and aldehydes in the vinegar-based kefir were also produced. The yield of acetic acid in the kefir vinegars was ∼79%. The acetic acid concentration was ∼41 g L−1, reaching the required standard for the Brazilian legislation accepts it as vinegar (4.0% acetic acid). Kefir vinegar showed good acceptance in the sensory analysis. The technology proposed here is novel by the application of immobilized-cell biomass (kefir grains) providing a mixed inocula and eliminating the use of centrifuge at the end of the fermentative process. This step will save energy demand and investment. This is the first study to produce apple vinegar using kefir grains.
doi:10.1016/j.bjm.2016.11.006
PMCID: PMC5498422  PMID: 28283415
Yeast; Saccharomyces; Lactobacillus; Apple; MALDI-TOF; Biospeckle Laser; Vinegar
10.  Sequencing-Based Analysis of the Bacterial and Fungal Composition of Kefir Grains and Milks from Multiple Sources 
PLoS ONE  2013;8(7):e69371.
Kefir is a fermented milk-based beverage to which a number of health-promoting properties have been attributed. The microbes responsible for the fermentation of milk to produce kefir consist of a complex association of bacteria and yeasts, bound within a polysaccharide matrix, known as the kefir grain. The consistency of this microbial population, and that present in the resultant beverage, has been the subject of a number of previous, almost exclusively culture-based, studies which have indicated differences depending on geographical location and culture conditions. However, culture-based identification studies are limited by virtue of only detecting species with the ability to grow on the specific medium used and thus culture-independent, molecular-based techniques offer the potential for a more comprehensive analysis of such communities. Here we describe a detailed investigation of the microbial population, both bacterial and fungal, of kefir, using high-throughput sequencing to analyse 25 kefir milks and associated grains sourced from 8 geographically distinct regions. This is the first occasion that this technology has been employed to investigate the fungal component of these populations or to reveal the microbial composition of such an extensive number of kefir grains or milks. As a result several genera and species not previously identified in kefir were revealed. Our analysis shows that the bacterial populations in kefir are dominated by 2 phyla, the Firmicutes and the Proteobacteria. It was also established that the fungal populations of kefir were dominated by the genera Kazachstania, Kluyveromyces and Naumovozyma, but that a variable sub-dominant population also exists.
doi:10.1371/journal.pone.0069371
PMCID: PMC3716650  PMID: 23894461
11.  Kefir treatment ameliorates dextran sulfate sodium-induced colitis in rats 
World Journal of Gastroenterology  2015;21(46):13020-13029.
AIM: To investigate the preventive effect of kefir on colitis induced with dextran sulfate sodium (DSS) in rats.
METHODS: Twenty-four male Wistar-albino rats were randomized into four groups: normal control, kefir-control, colitis, and kefir-colitis groups. Rats in the normal and kefir-control groups were administered tap water as drinking water for 14 d. Rats in the colitis and kefir-colitis groups were administered a 3% DSS solution as drinking water for 8-14 d to induce colitis. Rats in the kefir-control and kefir-colitis groups were administered 5 mL kefir once a day for 14 d while rats in the normal control and colitis group were administered an identical volume of the placebo (skim milk) using an orogastric feeding tube. Clinical colitis was evaluated with reference to the disease activity index (DAI), based on daily weight loss, stool consistency, and presence of bleeding in feces. Rats were sacrificed on the 15th day, blood specimens were collected, and colon tissues were rapidly removed. Levels of myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, interleukin (IL)-10, malondialdehyde, and inducible nitric oxide synthase (iNOS) were measured in colon tissue.
RESULTS: The DAI was lower in the kefir-colitis group than in the colitis group (on the 3rd and 5th days of colitis induction; P < 0.01). The DAI was also significantly higher in the colitis group between days 2 and 6 of colitis induction when compared to the normal control and kefir-control groups. The DAI was statistically higher only on the 6th day in the kefir-colitis group when compared to that in the normal control groups. Increased colon weight and decreased colon length were observed in colitis-induced rats. Mean colon length in the colitis group was significantly shorter than that of the kefir-control group. Kefir treatment significantly decreased histologic colitis scores (P < 0.05). MPO activity in the colitis group was significantly higher than in the kefir-control group (P < 0.05). Kefir treatment significantly reduced the DSS colitis-induced TNF-α increase (P < 0.01). No statistically significant differences were observed among groups for IL-10 and MDA levels. Colon tissue iNOS levels in the colitis group were significantly higher than those in the control and kefir-colitis groups (P < 0.05).
CONCLUSION: Kefir reduces the clinical DAI and histologic colitis scores in a DSS-induced colitis model, possibly via reduction of MPO, TNF-α, and iNOS levels.
doi:10.3748/wjg.v21.i46.13020
PMCID: PMC4674720  PMID: 26676086
Colitis; Dextran sulfate sodium; Inflammatory bowel disease; Kefir; Probiotic
12.  Characterization of pomegranate juice and whey based novel beverage fermented by kefir grains 
Mixture of pomegranate juice and whey was evaluated as a potential substrate for production of a novel probiotic beverage by kefir grains. Different fermentation conditions were used as viz: two fermentation temperature (19 ºC and 25 ºC) and two levels of kefir grains inoculum (5 % and 8%w/v). pH, acidity, lactose consumption as well as organic acids formation were determined during 32 hours of fermentation. Results showed that kefir grains were able to utilize lactose and decrease pH, increase acidity, produce lactic acid and acetic acid, while the level of citric acid decreased. It was observed these change depended on temperature and level of kefir grains with the highest changes at the temperature of 25 ºC and kefir grains inoculum of 8%w/v. Pomegranate juice and whey mixture therefore may serve as a suitable substrate for the production of novel probiotic dairy-fruit juice beverage by kefir grains and the sensory characteristics of this beverage were shown desirable results.
doi:10.1007/s13197-014-1412-9
PMCID: PMC4444931  PMID: 26028755
Kefir; Fermentation; Pomegranate juice; Whey
13.  Pharmacodynamics of Anidulafungin against Clinical Aspergillus fumigatus Isolates in a Nonneutropenic Murine Model of Disseminated Aspergillosis 
Azole resistance is an emerging increasing problem in Aspergillus fumigatus that results in treatment failure. Alternative treatments may improve the therapeutic outcome in patients with azole-resistant invasive aspergillosis (IA). Little is known about the in vivo efficacy of the echinocandin anidulafungin (AFG) in IA. The in vivo efficacy of 2.5, 5, 10, and 20 mg/kg of body weight AFG was assessed against two clinical Aspergillus fumigatus isolates with identical AFG minimum effective concentrations (MECs; 0.03 mg/liter) in a murine model of IA: a wild-type voriconazole (VCZ)-susceptible (VCZs) A. fumigatus isolate (AZN 8196) and a VCZ-resistant (VCZr) A. fumigatus isolate (V52-35) harboring the TR34/L98H resistance mechanism (substitution at codon L98 in combination with a 34-bp tandem repeat in the promoter region of the CYP51A gene). The pharmacokinetics of AFG were also assessed for each dose. Increasing doses increased survival for both isolates in a manner dependent on the AFG dose level (R2 = 0.99 and 0.95, respectively) up to a maximum of 72.7% and 45.45% for the VCZs and VCZr isolates, respectively. The area under the concentration-time curve (AUC) correlated significantly with the dose in a linear fashion over the entire dosing range (R2 = 0.86). The Hill equation with a variable slope fitted the relationship between the 24-h AUC/MEC ratio and 14-day survival well (R2 = 0.87; P < 0.05). The 50% effective AUC/MEC for total AFG was 126.5 (95% confidence interval, 79.09 to 202.03). AFG treatment improved the survival of mice in a dose-dependent manner; however, a maximal response was not achieved with either isolate even in those treated with the highest AFG dose.
doi:10.1128/AAC.01430-12
PMCID: PMC3535895  PMID: 23114773
14.  Control of Aflatoxin Production of Aspergillus flavus and Aspergillus parasiticus Using RNA Silencing Technology by Targeting aflD (nor-1) Gene 
Toxins  2011;3(6):647-659.
Aspergillus flavus and Aspergillus parasiticus are important pathogens of cotton, corn, peanuts and other oil-seed crops, producing toxins both in the field and during storage. We have designed three siRNA sequences (Nor-Ia, Nor-Ib, Nor-Ic) to target the mRNA sequence of the aflD gene to examine the potential for using RNA silencing technology to control aflatoxin production. Thus, the effect of siRNAs targeting of two key genes in the aflatoxin biosynthetic pathway, aflD (structural) and aflR (regulatory gene) and on aflatoxin B1 (AFB1), and aflatoxin G1 (AFG1) production was examined. The study showed that Nor-Ib gave a significant decrease in aflD mRNA, aflR mRNA abundance, and AFB1 production (98, 97 and 97% when compared to the controls) in A. flavus NRRL3357, respectively. Reduction in aflD and aflR mRNA abundance and AFB1 production increased with concentration of siRNA tested. There was a significant inhibition in aflD and AFB1 production by A. flavus EGP9 and AFG1 production by A. parasiticus NRRL 13005. However, there was no significant decrease in AFG1 production by A. parasiticus SSWT 2999. Changes in AFB1 production in relation to mRNA levels of aflD showed a good correlation (R = 0.88; P = 0.00001); changes in aflR mRNA level in relation to mRNA level of aflD also showed good correlation (R = 0.82; P = 0.0001). The correlations between changes in aflR and aflD gene expression suggests a strong relationship between these structural and regulatory genes, and that aflD could be used as a target gene to develop efficient means for aflatoxin control using RNA silencing technology.
doi:10.3390/toxins3060647
PMCID: PMC3202845  PMID: 22069731
siRNA; aflD (nor-1) gene; aflR gene; aflatoxin; real-time PCR
15.  Chronic administration of the probiotic kefir improves the endothelial function in spontaneously hypertensive rats 
Background
The beverage obtained by fermentation of milk with kefir grains, a complex matrix containing acid bacteria and yeasts, has been shown to have beneficial effects in various diseases. However, its effects on hypertension and endothelial dysfunction are not yet clear. In this study, we evaluated the effects of kefir on endothelial cells and vascular responsiveness in spontaneously hypertensive rats (SHR).
Methods
SHR were treated with kefir (0.3 mL/100 g body weight) for 7, 15, 30 and 60 days and compared with non-treated SHR and with normotensive Wistar-Kyoto rats. Vascular endothelial function was evaluated in aortic rings through the relaxation response to acetylcholine (ACh). The balance between reactive oxygen species (ROS) and nitric oxide (NO) synthase was evaluated through specific blockers in the ACh-induced responses and through flow cytometry in vascular tissue.
Results
Significant effects of kefir were observed only after treatment for 60 days. The high blood pressure and tachycardia exhibited by the SHR were attenuated by approximately 15 % in the SHR-kefir group. The impaired ACh-induced relaxation of the aortic rings observed in the SHR (37 ± 4 %, compared to the Wistar rats: 74 ± 5 %), was significantly attenuated in the SHR group chronically treated with kefir (52 ± 4 %). The difference in the area under the curve between before and after the NADPH oxidase blockade or NO synthase blockade of aortic rings from SHR were of approximately +90 and −60 %, respectively, when compared with Wistar rats. In the aortic rings from the SHR-kefir group, these values were reduced to +50 and −40 %, respectively. Flow cytometric analysis of aortic endothelial cells revealed increased ROS production and decreased NO bioavailability in the SHR, which were significantly attenuated by the treatment with kefir. Scanning electronic microscopy showed vascular endothelial surface injury in SHR, which was partially protected following administration of kefir for 60 days. In addition, the recruitment of endothelial progenitor cells was decreased in the non-treated SHR and partially restored by kefir treatment.
Conclusions
Kefir treatment for 60 days was able to improve the endothelial function in SHR by partially restoring the ROS/NO imbalance and the endothelial architecture due to endothelial progenitor cells recruitment.
doi:10.1186/s12967-015-0759-7
PMCID: PMC4696190  PMID: 26715471
Kefir; Probiotics; Spontaneously hypertensive rat; Oxidative stress; Endothelial dysfunction
16.  Mycoflora and Co-Occurrence of Fumonisins and Aflatoxins in Freshly Harvested Corn in Different Regions of Brazil 
Natural mycoflora and co-occurrence of fumonisins (FB1, FB2) and aflatoxins (AFB1, AFB2, AFG1 and AFG2) in freshly harvested corn grain samples from four regions of Brazil were investigated. Fusarium verticillioides was predominant in all samples. Analysis of fumonisins showed that 98% of the samples were contaminated with FB1 and 74.5% with FB1 + FB2, with toxin levels ranging from 0.015 to 9.67 μg/g for FB1 and from 0.015 to 3.16 μg/g for FB2. Twenty-one (10.5%) samples were contaminated with AFB1, seven (3.5%) with AFB2 and only one (0.5%) with AFG1 and AFG2 Co-contamination with aflatoxins and fumonisins was observed in 7% of the samples. The highest contamination of fumonisins and aflatoxins was observed in Nova Odessa (SP) and Várzea Grande (MT), respectively. The lowest contamination of these mycotoxins was found in Várzea Grande and Nova Odessa, respectively.
doi:10.3390/ijms10115090
PMCID: PMC2808024  PMID: 20087478
fumonisins; aflatoxins; corn (Zea mays L.); mycotoxins; mycoflora; Fusarium verticillioides; Aspergillus flavus
17.  Microbial Succession and Flavor Production in the Fermented Dairy Beverage Kefir 
mSystems  2016;1(5):e00052-16.
Traditional fermented foods represent relatively low-complexity microbial environments that can be used as model microbial communities to understand how microbes interact in natural environments. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein. In the process, the link between individual species, and associated pathways, with flavor compounds is revealed and several genes that could be responsible for the purported gut health-associated benefits of consuming kefir are identified. Ultimately, in addition to providing an important fundamental insight into microbial interactions, this information can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods.
ABSTRACT
Kefir is a putatively health-promoting dairy beverage that is produced when a kefir grain, consisting of a consortium of microorganisms, is added to milk to initiate a natural fermentation. Here, a detailed analysis was carried out to determine how the microbial population, gene content, and flavor of three kefirs from distinct geographic locations change over the course of 24-h fermentations. Metagenomic sequencing revealed that Lactobacillus kefiranofaciens was the dominant bacterial species in kefir during early stages of fermentations but that Leuconostoc mesenteroides became more prevalent in later stages. This pattern is consistent with an observation that genes involved in aromatic amino acid biosynthesis were absent from L. kefiranofaciens but were present in L. mesenteroides. Additionally, these shifts in the microbial community structure, and associated pathways, corresponded to changes in the levels of volatile compounds. Specifically, Acetobacter spp. correlated with acetic acid; Lactobacillus spp. correlated with carboxylic acids, esters and ketones; Leuconostoc spp. correlated with acetic acid and 2,3-butanedione; and Saccharomyces spp. correlated with esters. The correlation data suggest a causal relationship between microbial taxa and flavor that is supported by observations that addition of L. kefiranofaciens NCFB 2797 increased the levels of esters and ketones whereas addition of L. mesenteroides 213M0 increased the levels of acetic acid and 2,3-butanedione. Finally, we detected genes associated with probiotic functionalities in the kefir microbiome. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein and can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods.
IMPORTANCE Traditional fermented foods represent relatively low-complexity microbial environments that can be used as model microbial communities to understand how microbes interact in natural environments. Our results illustrate the dynamic nature of kefir fermentations and microbial succession patterns therein. In the process, the link between individual species, and associated pathways, with flavor compounds is revealed and several genes that could be responsible for the purported gut health-associated benefits of consuming kefir are identified. Ultimately, in addition to providing an important fundamental insight into microbial interactions, this information can be applied to optimize the fermentation processes, flavors, and health-related attributes of this and other fermented foods.
doi:10.1128/mSystems.00052-16
PMCID: PMC5080400  PMID: 27822552
dairy; flavor; kefir; metagenomics; microbiota
18.  Simultaneous detection of multiple mycotoxins in broiler feeds using a liquid chromatography tandem-mass spectrometry 
Mycotoxins are secondary fungal metabolites that are typically present in grain and feed ingredients used for animal feeds. An analytical method using LC-ESI-MS/MS was developed to quantify nine mycotoxins, consisting of aflatoxin B1 (AFB1), AFB2, AFG1, AFG2, T-2 toxin, deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEA) and ochratoxin A (OTA) in broiler feeds. In total, 100 samples of broiler feeds were collected from poultry farms in Central Thailand. The survey found that AFB1 and ZEA were the most prevalent mycotoxins in the feed samples at percentages of 93% and 63%, respectively. The limit of detections (LODs) of investigated mycotoxins was 0.20–0.78 ng/g. AFB2, DON, AFG1, NIV and T-2 toxin were also detectable at low contamination levels with percentages of 20%, 9%, 7%, 5% and 1%, respectively, whereas OTA and AFG2 were not detected in any of the feed samples. These results suggest that there is a very low level of risk of the exposure to mycotoxins in feeds obtained from broiler farms in Central Thailand.
doi:10.1292/jvms.15-0317
PMCID: PMC4785115  PMID: 26477362
broiler feed; LC-ESI-MS/MS; mycotoxin contamination
19.  Fine Structure of Tibetan Kefir Grains and Their Yeast Distribution, Diversity, and Shift 
PLoS ONE  2014;9(6):e101387.
Tibetan kefir grains (TKGs), a kind of natural starter for fermented milk in Tibet, China, host various microorganisms of lactic acid bacteria, yeasts, and occasionally acetic acid bacteria in a polysaccharide/protein matrix. In the present study, the fine structure of TKGs was studied to shed light on this unusual symbiosis with stereomicroscopy and thin sections. The results reveal that TKGs consist of numerous small grain units, which are characterized by a hollow globular structure with a diameter between 2.0 and 9.0 mm and a wall thickness of approximately 200 µm. A polyhedron-like net structure, formed mainly by the bacteria, was observed in the wall of the grain units, which has not been reported previously to our knowledge. Towards the inside of the grain unit, the polyhedron-like net structures became gradually larger in diameter and fewer in number. Such fine structures may play a crucial role in the stability of the grains. Subsequently, the distribution, diversity, and shift of yeasts in TKGs were investigated based on thin section, scanning electron microscopy, cloning and sequencing of D1/D2 of the 26S rRNA gene, real-time quantitative PCR, and in situ hybridization with specific fluorescence-labeled oligonucleotide probes. These show that (i) yeasts appear to localize on the outer surface of the grains and grow normally together to form colonies embedded in the bacterial community; (ii) the diversity of yeasts is relatively low on genus level with three dominant species – Saccharomyces cerevisiae, Kluyveromyces marxianus, and Yarrowia lipolytica; (iii) S. cerevisiae is the stable predominant yeast species, while the composition of Kluyveromyces and Yarrowia are subject to change over time. Our results indicate that TKGs are relatively stable in structure, and culture conditions to some extent shape the microbial community and interaction in kefir grains. These findings pave the way for further study of the specific symbiotic associations between S. cerevisiae and Lactobacillus bacteria in TKGs.
doi:10.1371/journal.pone.0101387
PMCID: PMC4076316  PMID: 24977409
20.  Total phenolic content and antioxidant activities of pomegranate juice and whey based novel beverage fermented by kefir grains 
Mixture of pomegranate juice and whey was evaluated as a potential substrate for production of a novel beverage by kefir grains. The effects of two different variables, fermentation, temperature (19 and 25 °C) and kefir grain amount (5 %w/v and 8 %w/v), on total phenolic content (TPC) and antioxidant activities of beverage were examined during a fermentation time of 32 h. TPC and antioxidant activities including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, reducing power, inhibition effect upon linoleic acid autoxidation and inhibition effect upon ascorbate autoxidation increased significantly (p < 0.05) during fermentation, but metal chelating effect showed no significant difference. The highest increases were observed when the temperature of 25 °C and kefir grain amount of 8 %w/v were applied. Results proved antioxidant activities of beverages were desirable and fermentation by kefir grains has the ability to enhance these antioxidant activities, as compared with unfermented beverage. Also pomegranate juice and whey were suitable media for producing a novel dairy-juice beverage.
doi:10.1007/s13197-015-2029-3
PMCID: PMC4711455  PMID: 26787994
Pomegranate juice; Whey; Kefir grain; Fermentation; Total phenolic content; Antioxidant activity
21.  The Neuroprotective Effect of Kefir on Spinal Cord Ischemia/Reperfusion Injury in Rats 
Objective
The main causes of spinal cord ischemia are a variety of vascular pathologies causing acute arterial occlusions. We investigated neuroprotective effects of kefir on spinal cord ischemia injury in rats.
Methods
Rats were divided into three groups : 1) sham operated control rats; 2) spinal cord ischemia group fed on a standard diet without kefir pretreatment; and 3) spinal cord ischemia group fed on a standard diet plus kefir. Spinal cord ischemia was performed by the infrarenal aorta cross-clamping model. The spinal cord was removed after the procedure. The biochemical and histopathological changes were observed within the samples. Functional assessment was performed for neurological deficit scores.
Results
The kefir group was compared with the ischemia group, a significant decrease in malondialdehyde levels was observed (p<0.05). Catalase and superoxide dismutase levels of the kefir group were significantly higher than ischemia group (p<0.05). In histopathological samples, the kefir group is compared with ischemia group, there was a significant decrease in numbers of dead and degenerated neurons (p<0.05). In immunohistochemical staining, hipoxia-inducible factor-1α and caspase 3 immunopositive neurons were significantly decreased in kefir group compared with ischemia group (p<0.05). The neurological deficit scores of kefir group were significantly higher than ischemia group at 24 h (p<0.05).
Conclusion
Our study revealed that kefir pretreatment in spinal cord ischemia/reperfusion reduced oxidative stress and neuronal degeneration as a neuroprotective agent. Ultrastructural studies are required in order for kefir to be developed as a promising therapeutic agent to be utilized for human spinal cord ischemia in the future.
doi:10.3340/jkns.2015.57.5.335
PMCID: PMC4479714  PMID: 26113960
Spinal cord ischemia; Cultured milk products; Reperfusion injury
22.  Monoclonal IgA Antibodies for Aflatoxin Immunoassays 
Toxins  2016;8(5):148.
Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort.
doi:10.3390/toxins8050148
PMCID: PMC4885063  PMID: 27187470
mycotoxin; monoclonal antibody; immunoglobulin A; orientation; immunoaffinitycolumn; ELISA
23.  Evaluation of Freeze-Dried Kefir Coculture as Starter in Feta-Type Cheese Production 
The use of freeze-dried kefir coculture as a starter in the production of feta-type cheese was investigated. Maturation of the produced cheese at 4°C was monitored for up to 70 days, and the effects of the starter culture, the salting method, and the ripening process on quality characteristics were studied. The use of kefir coculture as a starter led to increased lactic acid concentrations and decreased pH values in the final product associated with significantly higher conversion rates compared to salted rennet cheese. Determination of bacterial diversity at the end of the ripening process in salted kefir and rennet cheeses by denaturing gradient gel electrophoresis technology, based on both DNA and RNA analyses, suggested a potential species-specific inhibition of members of the genera Staphylococcus and Psychrobacter by kefir coculture. The main active microbial associations in salted kefir cheese appeared to be members of the genera Pseudomonas and Lactococcus, while in salted rennet cheese, Oxalobacteraceae, Janthinobacterium, Psychrobacter, and Pseudomonas species were noted. The effect of the starter culture on the production of aroma-related compounds responsible for cheese flavor was also studied by the solid-phase microextraction-gas chromatography-mass spectrometry technique. Kefir coculture also appeared to extend the shelf life of unsalted cheese. Spoilage of kefir cheese was observed on the 9th and 20th days of preservation at 10 and 5°C, respectively, while spoilage in the corresponding rennet cheese was detected on the 7th and 16th days. Microbial counts during preservation of both types of unsalted cheese increased steadily and reached similar levels, with the exception of staphylococci, which were significantly lower in unsalted kefir cheese. All types of cheese produced with kefir as a starter were approved and accepted by the panel during the preliminary sensory evaluation compared to commercial feta-type cheese.
doi:10.1128/AEM.03078-05
PMCID: PMC1563647  PMID: 16957238
24.  Aflatoxin Regulations and Global Pistachio Trade: Insights from Social Network Analysis 
PLoS ONE  2014;9(3):e92149.
Aflatoxins, carcinogenic toxins produced by Aspergillus fungi, contaminate maize, peanuts, and tree nuts in many regions of the world. Pistachios are the main source of human dietary aflatoxins from tree nuts worldwide. Over 120 countries have regulations for maximum allowable aflatoxin levels in food commodities. We developed social network models to analyze the association between nations’ aflatoxin regulations and global trade patterns of pistachios from 1996–2010. The main pistachio producing countries are Iran and the United States (US), which together contribute to nearly 75% of the total global pistachio market. Over this time period, during which many nations developed or changed their aflatoxin regulations in pistachios, global pistachio trade patterns changed; with the US increasingly exporting to countries with stricter aflatoxin standards. The US pistachio crop has had consistently lower levels of aflatoxin than the Iranian crop over this same time period. As similar trading patterns have also been documented in maize, public health may be affected if countries without aflatoxin regulations, or with more relaxed regulations, continually import crops with higher aflatoxin contamination. Unlike the previous studies on maize, this analysis includes a dynamic element, examining how trade patterns change over time with introduction or adjustment of aflatoxin regulations.
doi:10.1371/journal.pone.0092149
PMCID: PMC3966772  PMID: 24670581
25.  Immobilized Saccharomyces Cerevisiae as a potential aflatoxin decontaminating agent in pistachio nuts 
ABSTRACT
In this study, we investigated the binding ability of Saccharomayces cerevisiae to aflatoxin in pistachio nuts. The obtained results indicate that S. cerevisiae has an aflatoxin surface binding ability of 40% and 70% (with initial aflatoxin concentrations of 10 and 20 ppb) in the exponential phase. Acid treatments increase this ability to approximately 60% and 73% for the two concentrations of aflatoxin, respectively. Heat treatments also enhance surface binding to 55% and 75%, respectively. Binding appears to be a physical phenomenon that saturates within the first 2–3 hours of the process. The obtained results indicate that yeast immobilization for toxin reduction on aflatoxin-contaminated pistachios had no effect on qualitative characteristics, such as color, texture, and peroxide value. Yeast cells, viable or nonviable, are effective for aflatoxin binding, and this property could lead to a promising solution to aflatoxin contamination in high-risk foods.
doi:10.1590/S1517-838220100001000014
PMCID: PMC3768613  PMID: 24031467
Aflatoxin; Pistachio nut; Saccharomayces cerevisiae; Surface binding

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