Apple Cider Research Articles

Inhibitory Potency of Chlorogenic Acid from Apple Cider Vinegar Against Alzheimer’s Disease: Molecular Docking and Dynamics Validation

Objective The primary cause of memory loss is Alzheimer’s disease (AD). Recent studies have shown that natural compounds like apple cider vinegar (ACV) have anti-Alzheimer’s capabilities. Essential components of ACV, such as gallic acid and chlorogenic acid, may be in charge of the drug’s pharmacological effects. Methods Using molecular docking and dynamics (MD), the current work looks at the aspect of ACV that protects against AD. To study the conformational relationships and interaction mechanisms between two biological molecules (such as interactions between proteins and drugs or between proteins), MD simulation is frequently used. MD can help understand molecular structural differences between proteins and small compounds. We used acetylcholinesterase (AChE, PDB ID: 1UT6) to MD chlorogenic and gallic acids, as well as the currently prescribed medication rivastigmine (Standard medication). Furthermore, we determine the binding affinity, which may be responsible for AChE inhibition. MD simulations were performed on docked complexes of chlorogenic acid, gallic acid, and rivastigmine with receptor 1UT6 for a 300 ns trajectory to ensure the stability of docked ligand-protein complexes. Results The results showed that chlorogenic acid has the highest binding affinity and stability for AChE inhibition. In the docking and dynamics analysis, both techniques have predicted chlorogenic acid to be a potential constituent of ACV which shows a similar activity when compared to rivastigmine by virtue of binding affinity. Conclusion These findings identify chlorogenic acid as the key component of ACV that protects against AD-related cognitive and behavioral impairments. This finding will be critical in the development of ACV-based drugs for Alzheimer’s disease treatment.

Anti‐mold properties of fermented apple juice in bread

AbstractBackground and ObjectivesBread may be spoiled by mold, which results in major economic losses. The aim of this study was to optimize the anti‐mold properties of apple juice fermented with Propionibacterium freudenreichii to get high levels of propionic acid with lesser amounts of acetic acid, which impairs bread taste.FindingsAfter fermentation for 7 days at 35°C, apple juice (half‐diluted to about 4% fermentable sugars; with 1% yeast extract; pH adjusted to 6.0) contained 1.0% propionic acid and little amounts of acetic acid (about 0.35%).ConclusionsIn bread, the anti‐mold activity of fermented apple juice (5%, fb) was similar to that of apple cider vinegar (2.44%, fb).Significance and NoveltyFermented apple juice is a novel mold inhibitor for bread that may replace apple cider vinegar and part of calcium propionate.

The impact of apple cider vinegar on nonalcoholic fatty liver disease in rainbow trout, Oncorhynchus mykiss: A study of therapeutic potential and health benefits

AbstractIn this study, we aimed to experimentally induce fatty liver disease in rainbow trout, Oncorhynchus mykiss, and then assessed the illness recovery process, growth, and changes in the expression of FAAH and ACADL genes in both healthy (0 [C2] and 4% apple cider vinegar [T4]) and diseased fish (0 [C1], 1 [T1], 2 [T2], and 4% [T3]) apple cider vinegar. To conduct the study, 180 rainbow trout were randomly assigned to six different experimental treatments, each with three replications. The investigation lasted for 60 days. Growth indices, liver histology, blood biochemical parameters, and transcription of the ACADL and FAAH genes in the liver tissue were measured. The study found no significant differences in the final weights across all the treatments. Apple cider vinegar (ACV) administration resulted in a decrease in AST, ALT, and ALP; however, these values did not show a significant difference from C2. In T3, triglycerides significantly decreased (p < 0.05), whereas in T4, triglycerides significantly increased (p < 0.05). Hepatocytes from ACV‐containing treatments showed reduced fat compared with T4 and the control group (C1). While there was a significant difference (p < 0.05) in the expression of the FAAH gene, there was no significant difference (p > 0.05) in the expression of the ACADL gene between experimental treatments. The findings of our study indicate that an inclusion of up to 2% ACV may have positive effects on trout aquaculture and NAFLD treatment.

Effects of Various Solutions on Color Stability and Surface Hardness of Nanohybrid Dental Composite Under Simulated Oral Conditions.

This study evaluated effects of various solutions on color stability and surface hardness of a nanohybrid dental composite in simulated oral environments. Sixty-four composite disks were fabricated and randomly allocated into eight groups (n = 8 per group): Artificial saliva (AS), Biotene (B), passion fruit juice (PFJ), orange juice (OJ), Sprite (S), Coca-Cola (CO), apple cider vinegar (ACV), and cranberry juice (CJ). Specimens were immersed in respective solutions at 37°C for 28 days. Surface microhardness was assessed using Vickers microhardness test, and color alterations were quantified using SpectroShade Micro spectrophotometer. Measurements were taken 24 hours after initial polymerization and after 28-day immersion period. Statistical analysis was performed using mixed model ANOVA. After 28 days, specimens exhibited significant changes in microhardness and color. Polished surfaces showed microhardness decreases of 21.9-35.5%, with ACV and CJ causing largest reductions. Non-polished surfaces unexpectedly showed increased microhardness (11.2-17.4%). Color changes were more pronounced on polished surfaces, with CO and CJ causing maximum alterations. Statistical analysis revealed significant interactions between surface treatment, staining media, and immersion time (p < 0.05). All experimental groups demonstrated significant changes, highlighting composite materials' susceptibility to environmental factors. Even well-polymerized and polished surfaces underwent alterations, emphasizing necessity for periodic follow-up and maintenance polishing in esthetic restorations. The present research emphasizes significance of oral environmental factors on composite restoration longevity and esthetics, advocating for patient education on dietary impacts and tailored maintenance strategies to preserve restoration quality. How to cite this article: Shetty P, Jayakumar HDN, Pulickal TJ, et al. Effects of Various Solutions on Color Stability and Surface Hardness of Nanohybrid Dental Composite Under Simulated Oral Conditions. J Contemp Dent Pract 2024;25(7):669-676.

Cider Terroir: Influence of Regionality on Australian Apple Cider Quality

Understanding how regionality influences the key characteristics of cider will assist the industry to produce a premium and consistent quality product. Three dessert apple varieties were sourced from three (minimum) orchards per region from nine major growing regions across Australia over two seasons. Ciders produced from these apples were analysed for base quality characteristics, including total phenolic content (TPC), pH, total soluble solids (TSS) and titratable acidity (TA). Across both seasons, region had a significant influence on TPC, with the region model explaining approximately 25% of the variation in TPC. TSS and TA were significantly influenced by an interaction between variety and region, with the model accounting for approximately 60% and 75% of the variation, respectively, over both seasons. An interaction between variety and region influenced pH results in the first season, with only mean effects in the second season. A climate model was developed using average rainfall data and growing degree days (GDDs). Differences in climate accounted for most, but not all, of the regional variation observed in cider quality measurements. These results demonstrate that the apple growing region can significantly impact the resulting cider quality. Such findings are critical for supporting cider producers to make informed decisions when sourcing fruit.

Microbiological studies on Escherichia coli and Klebsiella pneumoniae causing vaginal and urinary tract inflammation; prevalence, antibiotics-resistance and natural products susceptibility

Great efforts have been made for controlling multidrug-resistant bacteria (MDR). The antibacterial activity of natural products is an effective strategy due to its beneficial effects on human health. This study focused on the isolation, identification, studying the prevalence and the susceptibility of Escherichia coli and Klebsiella spp. to some natural products. After isolation, the isolates primarily identified using biochemical and API tests. Then, the antibiotic susceptibility test was done to reveal the most antibiotics resistant isolates before further identification of Escherichia coli and Klebsiella pneumoniae using phoA and gyrA genes, respectively. Furthermore, the sensitivity of the most antibiotics resistant isolates to apple cider vinegar (ACV), garlic oil (GO), coconut oil (CNO), tea tree oil (TTO), and lavender oil (LO) was estimated. All tested extracts especially ACV and TTO showed good antibacterial activity against MRD selected isolates. Statistical analysis indicated that there was significant difference in ACV and TTO antibacterial response between Escherichia coli and Klebsiella pneumoniae whereas there was no significant difference between natural products activity when tested against Escherichia coli and Klebsiella pneumoniae seperatly. GC-MS spectroscopy analysis revealed the most prominent active constituents present in tested ACV and TTO. Analyzing the 16S rRNA sequence confirmed the two most MRD pathogenic isolates that showed sensitivity to ACV and TTO were Escherichia coli MS1 and Klebsiella pneumoniae MS47, respectively. Essential oils tea tree and apple cider vinegar showed good antibacterial activities against antibiotics-resistant Escherichia coli and Klebsiella pneumoniae causing vaginal and urinary tract inflammation.

Effect of trap bait and colours, and number of entry holes in monitoring of Drosophilidae (Diptera) species in a fig orchard

The study's objective was to evaluate how the capture rate of bottle traps is influenced by their color, number of entry holes, and bait liquids.For this purpose, yellow, blue, green, red, black, white, and transparent (colourless) traps were tested. Apple cider vinegar, grape vinegar, white wine, yeast, and water (control) were used as bait liquids. The number of entry holes was 2, 4, 6, and 8 holes per trap on the side of the bottle traps. The experiments were conducted in a randomised block design with three replicates in a fig orchard (variety Bursa Siyahı) with 437 trees in Aydın province from September 2018 to March 2019. In total, 48 traps were mounted on the experimental trees (1 trap per 1 tree), and counting of the drosophilid individuals in the traps was performed weekly. Red-coloured traps attracted the highest number of drosophilid individuals, followed by yellow-coloured. Regarding different baits, grape vinegar attracted the most drosophilid individuals, followed by white wine. Concerning the number of trap entry holes, the highest number of individuals were caught in traps with the highest number of holes, 8. In all traps, Zaprionus tuberculatus Malloch, 1932 was the species caught in the highest numbers, followed by Drosophila subobscura Collin, 1936 and Drosophila suzukii (Matsumura, 1931). Z. tuberculatus was mostly caught in traps containing grape vinegar, while D. subobscura and D. suzukii were mainly caught in traps containing white wine. In our study, the red-coloured traps containing vinegar with 8 entry holes were the most effective in monitoring drosophilid populations.

The influence of ascorbic and sulfurous acids on the intensity of oxidative processes in apple cider

The influence of ascorbic and sulfurous acids on the intensity of oxidative processes in apple cider

Apple cider vinegar in folk and modern medicine: a historical review and current scientific evidence.

Introduction and Aim of the Study: Apple cider vinegar (ACV) has been used for centuries in various cultural and medical contexts globally. This study seeks to explore ACV's traditional and contemporary applications, evaluating scientific evidence for its efficacy and safety. Material and Methods: This review synthesizes information from PubMed, Google Scholar, and other scientific sources. Key search terms included "apple cider vinegar," "apple cider vinegar medical use," "apple cider vinegar antimicrobial effect," "apple cider vinegar lipid profile," "apple cider vinegar glucose level," "apple cider vinegar preparation," and "apple cider vinegar historical use." Results: Research supports ACV's traditional role as a disinfectant, demonstrating effectiveness against a range of bacteria and fungi. Both animal and human studies suggest ACV may help lower cholesterol and triglyceride levels while boosting HDL cholesterol. Evidence indicates ACV can enhance insulin sensitivity and reduce blood glucose levels, offering benefits for type 2 diabetes management. ACV shows antioxidant properties, potential for lowering blood pressure, and use in certain skin diseases, though further research is needed to confirm these effects. Conclusions: The historical use of ACV in traditional medicine finds partial support in modern scientific research. Its antimicrobial, lipid-modulating, and glucose-regulating properties are documented, suggesting potential benefits for cardiovascular health, weight management, and metabolic regulation. Nonetheless, the evidence for its cosmetic applications and impact on muscle cramps remains less definitive. Additional research involving larger, diverse populations is necessary to fully validate ACV's benefits and establish safe usage guidelines.

Synergistic Amelioration of Letrozole-induced Polycystic Ovary Syndrome in Rats: A Therapeutic Approach with Apple Cider Vinegar and Metformin Combination.

The present study was conducted to evaluate the combination effect of apple cider vinegar (ACV) and metformin against letrozole-induced polycystic ovary syndrome (PCOS).Female Wistar rats were administered letrozole (1mg/kg/day, p.o) for 21 days, except for the control group of animals. On the 22nd day, PCOS-induced animals were segregated into 4 groups and administered with CMC, ACV, metformin, and a combination of ACV and metformin, respectively. The treatments were continued for 15 days, and on the 36th day, all the animals were sacrificed for biochemical (blood glucose, lipid profile), hormonal (sex hormones and adiponectin), and pro-inflammatory mediator estimations in blood samples. The ovarian tissue samples were used for oxidative stress parameters and histological alterations.The PCOS control animals showed a significant alteration in the estrous cycle. The administration of letrozole resulted in the alteration of hormonal balance and elevation of body weights, glycemic state, lipid profile, pro-inflammatory mediators in serum, and oxidative stress in ovarian samples. Individual treatment groups and combination treatment groups reversed the letrozole-induced alterations in PCOS animals, and more promising results were observed with combination therapy than with individual treatment groups. Further, the therapeutic potential of the combination treatment group was also confirmed by the histological observations in the ovarian samples.The study showed that the combination of ACV and metformin significantly alleviated letrozole-induced PCOS complications in rats. This might have been achieved by mitigating the hormonal imbalance, pro-inflammatory, hyperglycemic, and hyperlipidemic states in serum, and oxidative stress in the ovary samples.

Physicochemical and textural properties of gummy candies prepared with fruit vinegar

Foods with additional functional value derived from natural sources are becoming more and more in demand these days, including confectionary products. Therefore, the study aimed to develop a gummy candy recipe, enriched with different fruit vinegars and determine the components' effect on the products' mineral, acid, sugar, antioxidant, and polyphenol content, and texture of the confectionery products with analytical methods. In terms of macroelement content, potassium was present in the largest amount in the tested candies (167.87 and 178.83 mg kg-1) and liquid ingredients (57,71–306.93 mg kg-1). In the case of microelements, iron (4.81, 4.79 mg kg-1) and boron (4.62, 4.49 mg kg-1) were significant in the products, iron is also found in gelatin in significant quantities (3.51 mg kg-1), however, boron came exclusively from fruit-derived components (1.02–9.40 mg kg-1). Among the tested organic acids, malic acid (21.67–46.12 mg g-1), and acetic acid (47.74–50.06 mg g-1) were significant in both the raw materials and products, while the fructose content (2.44–439.35 mg g-1) was prominent among the sugars, but the glucose (75.99–163.44 mg g-1) and sucrose (73.61–157.00 mg g-1) content were also significant. Unfortunately, the polyphenol (1.37–1.46 mg GAE/g) and antioxidant (0.29–0.75 mg AAE/g) content of the products was not significant, maybe because of their heat sensitivity. In terms of the texture of the candies, the two candies prepared with different fruit vinegars had similar textural properties, however, in terms of hardness (3523.10 N/m2), gumminess (3266.25 N/m2), and chewiness (3425.33 N/m2) the gummy candy with apple cider vinegar proved to be better. Unlike many other confectionery products, the sugar content of the developed product is provided only by the used the apple concentrate, it does not contain added sugar or sweeteners. The addition of fruit vinegars has been proven to have a positive effect on the nutritional values of candies. By replacing gelatin with another gelling agent, the product can also be prepared in a vegan form.

Apple Cider Vinegar in the Combat Against Type 2 Diabetes and Obesity – An Overview of Recent Research

ABSTRACT Introduction Apple Cider Vinegar, primarily composed of acetic acid, is gaining more recognition for its possible health advantages, especially its anti-diabetic effects and its beneficial impact on weight loss. Aim of this study The aim of this review is to discuss the current state of knowledge regarding the influence of ACV on glycemic indices and weight management in diabetic and obese patients, explore the possibilities of preventing comorbidities, and assess the safety of ACV consumption. Materials and Methods Research was performed based on Pubmed and Google Scholar databases. The literature was reviewed using the keywords : Apple Cider Vinegar, Diabetes, Glucose, Obesity. Results Studies have confirmed that apple cider vinegar exhibits significant anti-obesity and anti-diabetic properties. It demonstrates dose- and time-dependent effects on reducing fasting glucose, postprandial glucose, glycated hemoglobin, and lipid parameters. It positively impacts weight loss, reducing waist and hip circumference, and enhances tissue sensitivity to insulin. Additionally, apple cider vinegar has been shown to potentiate the effects of metformin, with liquid consumption yielding better results than pill form. Conclusions Although initial findings are encouraging, additional research with a larger participant pool and an extended duration of vinegar consumption is necessary. The brevity of the study period restricts the observation of long-term effects, and a larger sample size would improve the applicability of the findings. Key words: apple cider vinegar; diabetes; glucose; obesity

Effect of Spraying with Soluamine L-24 and Apple Cider Vinegar on some Growth Traits of Le Conte Pear Seedlings Grafted onto Newly Planted Wild Pear Rootstock

Effect of Spraying with Soluamine L-24 and Apple Cider Vinegar on some Growth Traits of Le Conte Pear Seedlings Grafted onto Newly Planted Wild Pear Rootstock

Flavonoid-Rich Fruit Intake in Midlife and Late-Life and Associations with Risk of Dementia: The Framingham Heart Study

BackgroundFruits are an important source of flavonoids, and greater intake of dietary flavonoids in older adults has been shown to be associated with decreased risk of dementia. It is unclear whether this relationship is similar or different between younger adults and older adults.ObjectivesWe examined for associations between midlife and late-life intake of flavonoid-rich fruits and incident dementia. We hypothesized that greater total cumulative intake of flavonoid-rich fruits in midlife and late-life adults would be associated with reduced risk of all-cause dementia.DesignLongitudinal, cohort study design.SettingFramingham Heart Study, which is a longitudinal, multi-generational community-based cohort based in Framingham, Massachusetts, USA.ParticipantsParticipants from the Framingham Heart Study Offspring cohort were included (n = 2,790) who attended the fifth core exam between 1991 to 1995, and were dementiafree and at least 45 years of age at that time, as well as had valid food frequency questionnaires from the fifth to ninth core exams.MeasurementsConsumption of fruits with high flavonoid content or are important contributors to overall flavonoid intake was collected via food frequency questionnaire. Flavonoid-rich fruits from the food frequency questionnaire included raisins or grapes, prunes, bananas, fresh apples or pears, apple juice or cider, oranges, orange juice, grapefruit, grapefruit juice, strawberries, blueberries, and peaches, apricots, or plums. Dementia ascertainment was based on a multidisciplinary consensus committee, and included all-cause dementia and Alzheimer’s disease dementia diagnoses based on research criteria. Cox models were used to examine associations between cumulative fruit intake and incident dementia, stratified by midlife (45–59 years; n = 1,642) and late-life (60–82 years; n = 1,148).ResultsGreater cumulative total fruit intake in midlife, but not late-life, was significantly associated with a 44% decreased risk of all-cause dementia (HR = 0.56; 95% CI = 0.32–0.98; p = 0.044). Decreased risk of all-cause dementia was also associated with higher intake of apples or pears in midlife and late-life, as well as higher intake of raisins or grapes in midlife only, and higher intake of oranges, grapefruit, blueberries, and peaches, apricots, or plums in late-life only.ConclusionsAmong participants from the Framingham Heart Study, greater overall consumption of flavonoid-rich fruits in midlife was associated with reduced risk of dementia, though intake of specific fruits in midlife and late-life may have a protective role against developing dementia. These findings may help to inform future recommendations on when dietary interventions may be most beneficial to healthy brain aging across the life course.

Evaluation of Antimicrobial Effect of Ginger, Apple Cider Vinegar against Streptococcus Mutans, Enterococcus Faecalis and Candida Albicans.

Oral infections are most frequently caused by three oral microorganisms: Streptococcus mutans (S. mutans), Enterococcus faecalis (E. faecalis), and Candida albicans (C. albicans). To evaluate antimicrobial potential of 10% ginger extract (GE) and apple vinegar against S. Mutans, E. faecalis, and C. albicans. The GE and apple cider vinegar (ACV) were tested for microbiological evaluation to identify the inhibitory zone using the agar disk diffusion test against S. mutans, C. albicans, and E. faecalis. The 10% ethanolic GE and ACV showed a maximum zone of inhibition zone against S. mutans, E. Faecalis, and C. albicans. The intragroup comparison was insignificant. Apple vinegar and 10% ethanolic GE were shown to have good antimicrobial properties against all three tested pathogens.

The Influence of Yeast Strain on the Chemical, Chromatic, and Sensory Characteristics of ‘Wodarz’ Apple Cider

A regionally developed and adapted dessert apple, ‘Wodarz’, was explored for its potential in apple cider production because of its consistent productivity when other apple cultivars have struggled with North Dakota’s climate. Due to the importance of yeast strain on the perceived quality of fermentation products, five commercial yeast strains, three wine yeasts (EC1118, Maurivin B, and 71B), and two cider yeasts (WLP775 and WY4766) were evaluated for their impact on the physicochemical properties, color, and sensory characteristics of ‘Wodarz’ cider. By assessing dynamic changes, such as spectral properties and sugar content, a comparison among yeasts was conducted across multiple dimensions. The lightness, chroma, and hue all showed variations throughout fermentation, though not across the final ciders. However, differences in the final color of the ciders were identified via ΔE calculations. Each yeast contributed different aromas and tastes to the final ciders. Among yeast strains, EC1118 had the strongest aroma intensity. Despite having subdued aroma intensity, 71B had strong acidity tastes and WLP775 had strong fruity tastes. Thus, our research suggests that yeast strains are an applicable factor in determining the final sensory attributes of local ‘Wodarz’ cider. This is the first report of fermentation outcomes using ‘Wodarz’ apples for cider. ‘Wodarz’ can be aromatically described using terms such as apple, honey, herbal, rose, and floral and fruit notes. The overall taste of ‘Wodarz’ cider is characterized by apple, honey, and rose notes followed by black pepper and grass.

Apple cider vinegar for weight management in Lebanese adolescents and young adults with overweight and obesity: a randomised, double-blind, placebo-controlled study

Background and aimsObesity and overweight have become significant health concerns worldwide, leading to an increased interest in finding natural remedies for weight reduction. One such remedy that has gained popularity...

Determination of Patulin in Apple Juice and Apple-Derived Products Using a Robotic Sample Preparation System and LC-APCI-MS/MS.

Patulin, a toxic mycotoxin, can contaminate apple-derived products. The FDA has established an action level of 50 ppb (ng/g) for patulin in apple juice and apple juice products. To effectively monitor this mycotoxin, there is a need for adequate analytical methods that can reliably and efficiently determine patulin levels. In this work, we developed an automated sample preparation workflow followed by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCI-MS/MS) detection to identify and quantify patulin in a single method, further expanding testing capabilities for monitoring patulin in foods compared to traditional optical methods. Using a robotic sample preparation system, apple juice, apple cider, apple puree, apple-based baby food, applesauce, fruit rolls, and fruit jam were fortified with 13C-patulin and extracted using dichloromethane (DCM) without human intervention, followed by an LC-APCI-MS/MS analysis in negative ionization mode. The method achieved a limit of quantification of 4.0 ng/g and linearity ranging from 2 to 1000 ng/mL (r2 > 0.99). Quantitation was performed with isotope dilution using 13C-patulin as an internal standard and solvent calibration standards. Average recoveries (relative standard deviations, RSD%) in seven spike matrices were 95% (9%) at 10 ng/g, 110% (5%) at 50 ng/g, 101% (7%) at 200 ng/g, and 104% (4%) at 1000 ng/g (n = 28). The ranges of within-matrix and between-matrix variability (RSD) were 3-8% and 4-9%, respectively. In incurred samples, the identity of patulin was further confirmed with a comparison of the information-dependent acquisition-enhanced product ion (IDA-EPI) MS/MS spectra to a reference standard. The metrological traceability of the patulin measurements in an incurred apple cider (21.1 ± 8.0 µg/g) and apple juice concentrate (56.6 ± 15.6 µg/g) was established using a certified reference material and calibration data to demonstrate data confidence intervals (k = 2, 95% confidence interval).

Citrate-buffered Yamanaka medium allows to produce high-yield bacterial nanocellulose in static culture using Komagataeibacter strains isolated from apple cider vinegar.

Bacterial nanocellulose (BNC) is a sustainable, renewable, and eco-friendly nanomaterial, which has gained great attentions in both academic and industrial fields. Two bacterial nanocellulose-producing strains (CVV and CVN) were isolated from apple vinegar sources, presenting high 16S rRNA gene sequence similarities (96%-98%) with Komagataeibacter species. The biofilm was characterized by scanning electron microscopy (SEM), revealing the presence of rod-shaped bacteria intricately embedded in the polymeric matrix composed of nanofibers of bacterial nanocellulose. FTIR spectrum and XRD pattern additionally confirmed the characteristic chemical structure associated with this material. The yields and productivities achieved during 10 days of fermentation were compared with Komagataeibacter xylinus ATCC 53524, resulting in low levels of BNC production. However, a remarkable increase in the BNC yield was achieved for CVV (690% increase) and CVN (750% increase) strains at day 6 of the fermentation upon adding 22mM citrate buffer into the medium. This effect is mainly attributed to the buffering capacity of the modified Yakamana medium, which allowed to maintain pH close to 4.0 until day 6, though in combination with additional factors including stimulation of the gluconeogenesis pathway and citrate assimilation as a carbon source. In addition, the productivities determined for both isolated strains (0.850 and 0.917gL-1 d-1) compare favorably to previous works, supporting current efforts to improve fermentation performance in static cultures and the feasibility of scaling-up BNC production in these systems.

Real-time PCR primers and probes for the detection of Shiga toxin genes, including novel subtypes

Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes.A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence.The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.