Lupine Publishers | Evaluation of the Physicochemical and Thermal Properties of Folic Acid: Influence of the Energy of Consciousness Healing Treatment

 Lupine Publishers | Scholarly journal of Food and Nutrition

Abstract

Folic acid is vitamin B9, which is a water-soluble vitamin that plays an important role in cell growth, production of DNA and replication. This study was aimed to determine the influence of the Trivedi Effect^® Consciousness Energy Healing Treatment on the various physicochemical and thermal properties of folic acid by using the different analytical techniques. The study was done by dividing the folic acid sample into the control and treated parts; in which, no treatment was given to control, while the treated part received the Consciousness Energy Healing Treatment remotely by a renowned Biofield Energy Healer, Dahryn Trivedi. The analysis indicated that the particle sizes of the treated sample were reduced by 3.80% (d10), 5.18% (d50), 6.33% (d90), and 4.79% {D (4,3)}, respectively, which resulted in the increase in surface area by 4.60% compared with the control sample. The PXRD analysis indicated the changes in the peak intensities of the characteristic peaks of the treated sample and their corresponding crystallite sizes in the range from -45.76% to 277.50% and -87.55% to 15.88%, respectively, in comparison to the control folic acid sample.

The treated sample also showed a significant reduction in the average crystallite size by 19.37% compared with the control sample. The weight loss of the treated sample was increased during thermal degradation by 9.11% and therefore, the decreased residual mass by 36.56%, in comparison to the untreated sample. The DSC analysis of the treated sample revealed the increase in melting and degradation temperature by 10.48% and 7.32%, respectively; while the latent heat of fusion and decomposition were reduced by 2.27% and 34.35%, respectively, compared to the untreated sample. The overall study indicated that the Trivedi Effect^®- Consciousness Energy Healing Treatment has the significant impact of on the folic acid, which might be used as a novel approach for introducing some new polymorph of folic acid and thereby improving its solubility, bioavailability, and melting and degradation profile in comparison to the untreated sample. Hence, the Trivedi Effect^® could be used as a useful approach in developing the nutraceutical/pharmaceutical formulations of folic acid with improved performance and efficacy.

Keywords:Folic Acid; Consciousness Energy Healing Treatment; The Trivedi Effect^®; PXRD; Particle Size; TGA; DSC

Introduction

Folic acid is a type of vitamin B (vitamin B9) that is known for its water-solubility within the body. Its natural active form is tetrahydrofolate i.e., its fully reduced form, in which it serves as a 1-carbon donor during the purines and thymidine synthesis and in the homocysteine to methionine remethylation cycle. Folate is considered essential for normal cell growth, DNA production, and replication. Previous studies reported its use by various enzymes as a co-factor such as a thymidylate synthase and in folate homeostasis such as the reduced folate carrier, folylpolyglutamate synthase [1,2]. Folates serves an important role of a single carbon donor in various synthesis processes within the body such as the synthesis of serine from glycine, nucleotides form purine precursors, as a methyl donor to create methyl cobalamin i.e., further used in homocysteine to methionine re-methylation, and indirect use in the synthesis of transfer RNA [3].

The use of folic acid is also evident in the cell development, metabolism of various specific biochemical reactions within the body, as well as the metabolism of some specific anticonvulsant drugs. Folic acid is known for its interrelationship with vitamin B12 and its deficiency may increase the risk of neural tube defects (NTDs) and hyperhomocystinemia, i.e., increased risk of cardiovascular disease and NTDs [4]. Thus, to prevent its deficiency, oral folates are given to the patients as supplements that are generally available in two forms, folic and folinic acid. Folinic acid administration seems advantageous as it bypasses the steps of deconjugation and reduction that are essential in folic acid metabolism. Folinic acid is also considered more metabolically active, thus it may boost the levels of the coenzyme forms of the vitamin in the case when folic acid has little or no impact.

The therapeutic uses of folic acid are that it could reduce the level of homocysteine in the body and therefore, reduces the occurrence of neural tube defects; protects against neoplasia in ulcerative colitis; prevents cervical dysplasia; helps in treating vitiligo; and may help in increasing the resistance of the gingiva to local irritants that reduces the inflammation. Besides, the folate deficiency may cause various neuropsychiatric diseases such as schizophrenia-like syndromes, dementia, insomnia, forgetfulness, irritability, endogenous depression, peripheral neuropathy, organic psychosis, myelopathy, and restless legs syndrome, etc. [5-8]. The physicochemical properties of any drug such as its solubility, melting point, partition coefficient, etc. play a crucial role in its ADME profile. Hence, various approaches have been used in this way to alter the physicochemical properties of a drug to enhance its efficacy and biological activities in the body [9]. In recent days, the Consciousness Energy Healing Treatment is such an approach that is used by various scientists to modify the properties of drugs in relation to improve their bioavailability [10- 13]. A human has the ability to harness energy from the universe and can transmit it to any living organism(s) or non-living object(s) around the globe.

The object or recipient always receives energy and responds in a useful way. This process is known as the Trivedi Effect^® - Biofield Energy Healing Treatment [14,15]. The concept of Biofield Energy Healing is currently used as an alternative integrative approach that is widely accepted due to its ability to improve the quality of life by correcting the root cause of the diseases [16-18]. In a similar manner, the Trivedi Effect^®- Consciousness Energy Healing Treatment has been reported for its beneficial impact in the field of antimicrobial activity [19-21], agriculture and productivity [22,23], biotechnology [24,25], nutraceuticals [26,27], cancer research [28], bone health [29], skin health [30], and for altering the properties of metals, chemicals, ceramics and polymers [31-33], etc. This study was aimed to establish the impact of the Trivedi Effect^® on the physicochemical and thermal properties of folic acid with the help of various analytical techniques.

Materials and Methods

Chemicals and Reagents

The primary test sample folic acid was purchased from Alfa Aesar, USA and remaining chemicals were purchased in India.

Consciousness Energy Healing Treatment Strategies

The folic acid sample used in the study was first divided into two parts and termed as the control and Biofield Energy Treated sample based on the treatment. The control sample did not receive the Biofield Energy Treatment, but the sample was treated with a “sham” healer. The sham healer did not have any knowledge about the Biofield Energy Treatment. Besides, the treated sample was received the Trivedi Effect^®-Consciousness Energy Healing Treatment by the renowned Biofield Energy Healer, Dahryn Trivedi, USA, with her unique energy transmission process under standard laboratory conditions for 3 minutes. After the treatment, both the samples were kept in sealed conditions and characterized using sophisticated analytical techniques.

Characterization

The particle size analysis (PSA) was performed using Malvern Mastersizer 2000, from the UK using the wet method [34,35]. The powder x-ray diffraction (PXRD) analysis of folic acid powder sample was performed with the help of Rigaku MiniFlex-II Desktop X-ray diffractometer (Japan) [36,37]. The average crystallite size of the folic acid samples was calculated from XRD data using the Scherrer’s formula (1)

Where G is the crystallite size in nm, k is the equipment constant, λ is the radiation wavelength, β is the full-width at half maximum, and θ is the Bragg angle [38].

The thermal gravimetric analysis/differential thermogravimetric analysis TGA/DTG thermograms of folic acid were obtained with the help of TGA Q50 TA instruments. Similarly, the differential scanning calorimetry (DSC) analysis of folic acid was performed with the help of DSC Q200, TA instruments [39]. The % change of the treated folic acid was calculated compared with the control sample using the following equation 2:

Results and Discussion

Particle Size Analysis (PSA)

The particle size analysis of the treated sample was done and compared with the results of the control sample (Table 1) to analyse the impact of the Biofield Energy Treatment on the particle size distribution of the folic acid. The analysis indicated the reduction in the particle size values of the treated sample by 3.80%, 5.18%, 6.33%, and 4.79% at d10, d50, d90, and D (4, 3), respectively, as compared to the control sample.

The reduced particle size after the Biofield Energy Treatment of the folic acid sample resulted in the increased surface area as the SSA of the treated sample was found to be 1.82m²/g that is increased by 4.60% in comparison to the control sample (1.74m²/g). The particle size distribution of any drug plays a vital role in its performance and efficacy within the body by directly affecting its solubility and bioavailability [9,40]. The scientists already have been using the approach of reducing the particle size of the drug in increasing the effective surface area and thereby the dissolution and solubility of the drug [41]. Thus, it is suggested that the treated folic acid sample might show better solubility, dissolution, and bioavailability profile when used in formulation development as compared to the untreated sample.

d₁₀, d₅₀, and d₉₀: diameter of the particles corresponding to 10%, 50%, and 90% of the cumulative distribution, D (4,3): the average mass-volume diameter, and SSA: the specific surface area.

The PXRD studies of the control and treated samples were done and the corresponding diffractograms are given in Figure 1. The diffractogram’s analysis indicated the crystalline nature of both the samples due to the presence of sharp and intense peaks in the given figure (Figure 1). The further analysis (Table 2) helps in determining any changes between the control and treated sample in terms of the Bragg’s angles of the peaks, their relative peak intensities and corresponding crystallite sizes of the characteristic peaks. The study indicated the significant changes in the Bragg’s angles of the peaks of the treated sample in comparison to the characteristic peaks present in the diffractogram of the control sample. Also, the treated folic acid sample showed changes in the relative peak intensities and corresponding crystallite sizes in the range from -45.76% to 277.50% and -87.55% to 15.88%, respectively, as compared to the control sample.

Besides, the average crystallite size of the treated folic acid (138.25nm) also showed major alteration as it was significantly decreased by 19.37% in comparison to the control sample (171.46nm). The remarkable changes in the crystalline structure and crystal morphology of drugs might occur due to the possible formation of a novel polymorphic form of folic acid [42,43] after the Biofield Energy Treatment. This presumption could be done based on the analysis that the peak intensities and crystallite sizes of the treated folic acid sample were altered after the Consciousness Energy Healing Treatment in comparison to the untreated sample. Moreover, the novel polymorphic form of the compound may show better bioavailability and drug efficacy profile [44]; thus, the treated folic acid might be more bioavailable and effective as compared to the untreated sample.

Thermal Gravimetric Analysis (TGA)/ Differential Thermogravimetric Analysis (DTG)

The thermal stability analysis of the control and treated folic acid samples was done with the help of TGA/DTG technique. The TGA thermograms of the control and treated samples are given in Figure 2. Further analysis was done to analyse the differences between the degradation profile of the control and the treated sample (Table 3). It revealed that the treated sample showed increased weight loss by 9.11% during the thermal degradation in comparison to the control sample. Such an increase in the weight loss signifies the decrease in the residue weight of the treated sample remaining after the degradation by 36.56%, compared to the control sample. Thus, it showed that the treated folic acid sample showed increased thermal degradation, compared to the control sample.

The DTG analysis of both the samples, i.e., the control and treated folic acid samples showed four peaks in the DTG thermograms (Figure 3), that represented the temperature at which maximum thermal degradation has taken place. The analysis revealed that the maximum thermal degradation temperatures (T_max) corresponding to 1st and 2nd peak in the treated sample was reduced by 0.75% and 2.07%, respectively; while it was increased by 3.71% and 1.78% for the 3rd and 4th peaks, respectively as compared to the control sample. Hence, it could be suggested that the thermal degradation of the treated sample was reduced at higher temperatures after the Biofield Energy Treatment in comparison to the control folic acid sample. Thus, the overall analysis indicated the alterations in the thermal degradation profile of the treated sample in comparison to the untreated sample.

Differential Scanning Calorimetry (DSC) Analysis

The DSC analysis of both the samples i.e., the control and treated folic acid sample helps in studying and analysing the differences in their thermal behaviour such as melting and crystallization temperature etc. [45]. The previous studies reported that when folic acid was heated, the “Glu” moiety will first break down at ~180°C, followed by the degradation of pterin and PABA moieties. Afterward, when the sample was further heated, it loses the amide and acid functionalities at ~195°C, and then the crystalline folic acid degraded above 200°C in the form of the amorphous form [46]. The DSC analysis of both the samples indicated the presence of two peaks in their respective DSC thermograms (Figure 4). The first peak observed in the thermograms of both the samples i.e., the control and treated sample was endothermic in nature and denote the melting of the folic acid samples. The analysis indicated the significant increase in the melting temperature of the treated sample by 10.48%, while the corresponding Δ_Hfusion was reduced by 2.27% as compared to the control sample (Table 4).

Moreover, the second peak observed in the thermograms of both the samples is exothermic in nature that might denote the sample degradation on further heating. It was observed that the treated sample showed a significant increase in the degradation temperature by 7.32% compared with the control folic acid sample; however, the ΔHdegradation was significantly reduced by 34.35% compared to the control sample (Table 4). The DSC analysis indicated the improved stability of the treated folic acid sample during heating, which might happen as a result of some possible alterations in the crystallization structure [47] after the Biofield Energy Treatment. Hence, it could be suggested that the treated folic acid sample might be more thermally stable compared with the untreated sample.

Conclusions

The Trivedi Effect^®-Consciousness Energy Healing Treatment has been known previously for its significant effect on the properties of various compounds. This study also concluded the impact of the Biofield Energy Treatment on the physicochemical and thermal properties of the folic acid sample. It revealed the remarkable alterations in the particle size distribution of the treated sample i.e., the reduced particle size at d10, d50, d90, and D (4,3) by 3.80%, 5.18%, 6.33%, and 4.79%, respectively in comparison to the particle sizes of the control sample. Furthermore, the treated folic acid sample showed an increased specific surface area by 4.60% due to the reduced particle sizes compared with the untreated sample. The PXRD studies indicated major changes in the relative intensities of the characteristic peaks of the treated sample’s diffractogram along with the corresponding crystallite sizes in the range from -45.76% to 277.50% and -87.55% to 15.88%, respectively, compared with the untreated sample.

Besides, the Biofield Energy Treatment might also alter the average crystallite size of the treated sample that was significantly reduced by 19.37% as compared to the control sample. The TGA analysis of the treated folic acid sample indicated the increased weight loss during the thermal degradation by 9.11%; therefore, the residue weight remaining after the degradation was observed to be decreased by 36.56%, compared to the untreated sample. The DSC analysis suggested the significant increase in the melting point and degradation temperature of the treated sample as it was observed to be increased by 10.48% and 7.32%, respectively, compared to the control sample. However, the enthalpy changes i.e., ΔHfusion and ΔHdegradation was reduced by 2.27% and 34.35%, respectively compared with the enthalpy changes of the control sample during the process.

The overall study showed that the Trivedi Effect^®- Consciousness Energy Healing Treatment might be used as a new approach that might form a novel polymorphic form of the folic acid with improved dissolution, solubility, and bioavailability along with increased thermal stability as compared to the untreated sample. Thus, the Trivedi Effect^® Treated folic acid could be presumed as more beneficial in the pharmaceutical/nutraceutical preparations for treating and preventing various disorders such as, schizophrenia-like syndromes, dementia, insomnia, forgetfulness, irritability, endogenous depression, peripheral neuropathy, organic psychosis, myelopathy, and restless legs syndrome, eye disease age-related macular degeneration (AMD), allergic diseases, sleep problems, osteoporosis, etc.

Acknowledgement

The authors are grateful to Central Leather Research Institute, SIPRA Lab. Ltd., Trivedi Science, Trivedi Global, Inc., Trivedi Testimonials, and Trivedi Master Wellness for their assistance and support during this work

https://lupinepublishers.com/food-and-nutri-journal/fulltext/evaluation-of-the-physicochemical-and-thermal-properties-of-folic-acid.ID.000122.php

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Lupine Publishers | Complementary and Alternative Medicine: Impact of Consciousness Energy Healing Treatment on the Cholecalciferol (Vitamin D3)

 Lupine Publishers | Scholarly Journal of Food and Nutrition

Abstract

Cholecalciferol is a steroid hormone (7-dehydroxycholesterol), which helps in the absorption of dietary minerals like zinc, calcium, magnesium, iron, and phosphate and also responsible for other biological activity. In this research study, the influence of the Trivedi Effect®-Consciousness Energy Healing Treatment on the physicochemical and thermal properties of cholecalciferol was evaluated using the modern analytical technique. The cholecalciferol test sample was divided into two parts. One of the test samples was considered as a control sample, which did not receive the Biofield Energy Treatment; whereas, the other part was treated with the Consciousness Energy Healing Treatment remotely by a well-known Biofield Energy Healer, Gopal Nayak and termed as a treated sample. The particle size values in the treated cholecalciferol powder sample were significantly decreased by 78.94% (d₁₀), 26.21% (d₅₀), 22.01% (d₉₀), and 29.04% {D (4,3)}; thus, the specific surface area of the treated cholecalciferol was significantly increased by 174.12% compared to the control sample. The XRD peak intensities and crystallite sizes of the treated cholecalciferol powder sample were significantly altered ranging from -44.37% to 370.49% and -74.48% to 91.52%, respectively; therefore, the average crystallite size of the treated cholecalciferol was significantly decreased by 36.21% compared to the control cholecalciferol. The latent heat of fusion of the treated cholecalciferol was significantly increased by 25.89% compared to the control cholecalciferol. The weight loss was decreased by 1.48%; whereas, the residue amount was significantly increased by 107.02% in the treated sample compared with the control sample. Thus, the Trivedi Effect®-Consciousness Energy Healing Treatment generated a new polymorphic form of cholecalciferol which might offer better solubility, absorption, bioavailability, and be thermally more stable compared with the control sample. Henceforth, the Consciousness Energy Healing Treated cholecalciferol would be more beneficial to maintain the overall quality of life and it would be more useful in designing novel nutraceutical/pharmaceutical formulations for the better therapeutic responses against deficiency of vitamin D, osteoporosis, rickets, cardiovascular diseases, diabetes mellitus, cancer, mental disorders, multiple sclerosis, etc.

Keywords: Complementary and Alternative Medicine; Vitamin D₃; The Trivedi Effect®; Consciousness Energy Healing Treatment; Particle size; Surface area; PXRD; DSC; TGA/DTG

Introduction

Vitamin D₃ (cholecalciferol or 7-dehydroxycholesterol) is a steroid hormone produced in the skin once exposed to ultraviolet light and also obtained from dietary sources. It is a fat-soluble vitamin helps in the absorption of the dietary minerals like zinc, calcium, magnesium, iron, and phosphate and responsible for other biological activity, i.e., maintain healthy immune, skeletal, cardiovascular, and reproductive systems [1-3]. The major dietary sources of vitamin D are the cod liver oil, fatty fish like salmon and tuna, milk, nutraceutical and pharmaceutical supplements [2]. It is used for the prevention and treatment of several diseases like hypovitaminosis D, rickets, osteoporosis, diabetes mellitus, cardiovascular diseases, mental disorders, infections, cancer, multiple sclerosis, etc. [4-6]. Cholecalciferol nutritional deficiency mostly under-diagnosed and under-treated is pandemic all over the world [7]. The 1,25-Dihydroxycholecalciferol is the biologically active form of cholecalciferol known as calcitriol [5- 7]. The overdosing of cholecalciferol can be the cause of vomiting, constipation, hypercalcemia, polyuria, polydipsia, insomnia, confusion, kidney stone, weakness, and mental retardation [1].

The bioavailability of cholecalciferol is very poor. Similarly, some of the other factors that directly affect the bioavailability of cholecalciferol are dietary fiber, genetic factors, and cholecalciferol status [8,9]. The stability, solubility, and bioavailability of cholecalciferol are the major concern for the storage and nutraceutical/pharmaceutical formulations point of views, as it is insoluble to water and sensitive to the light and air [10,11]. The pharmaceutical and nutraceutical scientists are working hard for the improvement of physicochemical properties of the compounds for better dissolution, absorption, and bioavailability in the body [12]. In this concern, the Consciousness Energy Healing Treatment (the Trivedi Effect®) has been proved experimentally that, it has a substantial impact on various physicochemical properties and also bioavailability of nutraceutical and pharmaceutical entities [13-16]. The Trivedi Effect® is natural and only proven scientific phenomenon in which a specialist can harness this intelligent energy from the “Universe” and transfer it anywhere on the planet through the possible mediation of neutrinos [17]. Around the body of every living organism an infinite and para-dimensional unique electromagnetic field exists created from the continuous moment of the charged particles like ions, cells, blood, etc. is called a “Biofield”. The Biofield Energy Therapies have been reported with significantly beneficial outcomes against various disease [18]. The National Center for Complementary and Alternative Medicine (NCCAM) and the National Institutes of Health (NIH) have recommend and included the Energy therapy under the Complementary and Alternative Medicine (CAM) along with other therapies, i.e., homeopathy, acupuncture, Ayurvedic medicine, naturopathy, acupressure, Tai Chi, Qi Gong, Reiki, hypnotherapy, etc. The CAM has been accepted by most of the USA population [19,20]. On the other hand, the Consciousness Energy Healing Treatment (the Trivedi Effect®) has gained popularity all over the world and reported with the substantial impact on the physicochemical and behavioural properties of metals, ceramics, polymer, organic compounds, microorganisms, cancer cells, crops, etc. [21-31]. In this study the impact of the Trivedi Effect®-Consciousness Energy Healing Treatment on the physicochemical and thermal properties of cholecalciferol was evaluated using particle size analysis (PSA), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), and differential thermogravimetric analysis (DTG)/ thermogravimetric analysis (TGA).

Materials and Methods

Chemicals and Reagents

The cholecalciferol powder sample (> 98%) was purchased from Sigma-Aldrich, India and reaming chemicals were of analytical grade purchased in India. Consciousness Energy Healing Treatment Strategies The test sample vitamin D₃ was equally divided into two parts. One part of the vitamin D₃ sample was received the Consciousness Energy Healing Treatment (the Trivedi Effect®) remotely provided standard laboratory conditions for 3 minutes by a well-known Biofield Energy Healer, Gopal Nayak, India, known as a Biofield Energy Treated sample. Though, the second part of vitamin D₃ sample did not receive the Consciousness Energy Healing Treatment but treated with a “sham” healer called as a control sample. The “sham” healer doesn’t know anything about the Biofield Energy Treatment. After the treatment, both the samples were kept in sealed conditions and characterized using modern analytical techniques.

Characterization

The PSA of vitamin D₃ was performed using Malvern Mastersizer 2000 (the UK) using the wet method [32,33]. The PXRD analysis of vitamin D₃ powder sample was performed with the help of Rigaku Mini Flex-II Desktop X-ray diffractometer (Japan) [34,35]. The crystallites size was calculated from XRD data using the Scherrer’s formula (1)

Where k is the equipment constant, G is the crystallite size in nm, λ is the radiation wavelength, β is the full-width at half maximum, and θ is the Bragg angle [36,37].

Similarly, the DSC analysis of vitamin D₃ was performed with the help of DSC Q200, TA instruments. The TGA/DTG thermograms of vitamin D₃ were obtained with the help of TGA Q50 TA instruments [32,33]. The % change in particle size, specific surface area, peak intensity, crystallite size, latent heat, melting point, weight loss and the maximum thermal temperature of the Biofield Energy Treated vitamin D₃ was calculated compared with the control vitamin D₃ using the following equation 2:

Results and Discussion

Particle Size Analysis (PSA)

The particle sizes of the control and Biofield Energy Treated cholecalciferol were analyzed, and the data are presented in Table 1. The particle size values in the treated cholecalciferol were significantly decreased by 78.94%, 26.21%, 22.01%, and 29.04% at d₁₀, d₅₀, d₉₀, and D (4,3), respectively compared to the control cholecalciferol. Therefore, the specific surface area of the Biofield Energy Treated cholecalciferol (0.0625 m2/g) was significantly increased by 174.12% compared to the control sample (0.0228 m2/g). The results indicated that the Trivedi Effect®-Consciousness Energy Healing Treatment supposed to act as an external force for breaking the larger particles of cholecalciferol to smaller one; hence increased the surface area. The particle properties (size, shape, and surface area) of a drug molecule have a significant impact on the solubility, absorption, bioavailability, and the therapeutic efficacy [12, 38]. As cholecalciferol is a lipophilic compound and the solubility of it is very poor in water accountable for the poor bioavailability in the body [8,9]. The Consciousness Energy Healing Treated cholecalciferol may show better solubility, absorption, and therapeutic efficacy in the body.

Powder X-Ray Diffraction (PXRD) Analysis

The PXRD diffractograms of both the samples showed sharp and intense peaks (Figure 1), indicated that both the samples were crystalline. The control and Biofield Energy Treated cholecalciferol samples showed the highest peak intensity at 2θ equal to 17.76° and 17.97° in the powder XRD diffractograms (Table 2). The peak intensities of the treated cholecalciferol were significantly altered ranging from -44.37% to 370.49% compared to the control sample. Similarly, the crystallite sizes of the treated cholecalciferol sample were significantly altered ranging from -74.48% to 91.52% compared to the control sample. Largely, the average crystallite size of the Biofield Energy Treated cholecalciferol (246.75 nm) was significantly decreased by 36.21% compared with the control sample (386.8 nm). The change in the peak intensity of the crystalline compound indicated the alterations in the crystal morphology [38]. The alterations in the powder XRD pattern provide the proof of polymorphic transitions [39,40]. The Trivedi Effect®-Consciousness Energy Healing Treatment assumed to be responsible for the new polymorphic form of cholecalciferol probably through the Consciousness Energy via neutrino oscillations [17]. The drug performance, i.e., bioavailability, therapeutic efficacy, and toxicity of the pharmaceutical compound would be different from the original one due to different polymorphic forms [41-43]. Hence, the Consciousness Energy Healing Treated sample would be more efficacious novel pharmaceutical/nutraceutical formulations for the treatment of cholecalciferol deficiency disease.

Differential Scanning Calorimetry (DSC) Analysis

The cholecalciferol control and treated samples showed the sharp endothermic peak in the thermograms (Figure 2). The literature data closely match the experimental results [42]. The melting point and latent heat of fusion (ΔH fusion) of the Biofield Energy Treated cholecalciferol were increased by 0.38% and 25.89%, respectively compared with the control sample (Table 3). The change in the latent heat of fusion can be directly related to the disrupted intrinsic molecular chain and the crystal structure [43]. As per the result the thermal stability of the Consciousness Energy Healing Treated cholecalciferol was improved compared to the thermal stability of the control sample.

Thermal Gravimetric Analysis (TGA) / Differential Thermogravimetric Analysis (DTG)

The control and Biofield Energy Treated samples showed one step of thermal degradation (Figure 3). The weight loss of the Biofield Energy Treated cholecalciferol was decreased by 1.48%; whereas, the residue amount was significantly increased by 107.02% compared to the control sample (Table 4). Similarly, the control and Biofield Energy Treated cholecalciferol showed one peak of maximum thermal degradation temperature (T_max) in the thermograms (Figure 4). The T_max of the treated sample (292.01°C) was increased by 0.74% compared to the control sample (289.87°C). Overall, thermal analysis data of cholecalciferol samples indicated that the thermal stability of the Biofield Energy Treated sample was increased compared with the control sample.

Conclusions

The Consciousness Energy Healing Treatment (the Trivedi Effect®) has shown the significant effects on the particle and crystallite size, surface area, peak intensity, and thermal behavior of cholecalciferol. The particle size values in the Biofield Energy Treated cholecalciferol powder sample were significantly decreased by 78.94% (d₁₀), 26.21% (d₅₀), 22.01% (d₉₀), and 29.04% {D (4,3)}; thus, the specific surface area of the Biofield Energy Treated cholecalciferol was significantly increased by 174.12% compared to the control cholecalciferol. The powder XRD peak intensities and crystallite sizes of the Biofield Energy Treated cholecalciferol powder sample were significantly altered ranging from -44.37% to 370.49% and -74.48% to 91.52%, respectively; therefore, the average crystallite size of the Biofield Energy Treated sample was significantly decreased by 36.21% compared with the control sample. The ΔH fusion of the Biofield Energy Treated cholecalciferol was significantly increased by 25.89% compared to the control cholecalciferol. The total weight loss was decreased by 1.48%; whereas, the residue amount was significantly increased by 107.02% in the Biofield Energy Treated sample compared with the control sample. Thus, the Trivedi Effect®-Consciousness Energy Healing Treatment generated a new polymorphic form of cholecalciferol which might offer better solubility, absorption, bioavailability, and be thermally more stable compared with the control sample. Henceforth, the Consciousness Energy Healing Treated cholecalciferol would be more beneficial to maintain the overall quality of life and it would be more useful in designing novel nutraceutical/pharmaceutical formulations for the better therapeutic responses against deficiency of vitamin D, osteoporosis, rickets, cardiovascular diseases, diabetes mellitus, cancer, mental disorders, multiple sclerosis, etc.

Acknowledgements

The authors are grateful to Central Leather Research Institute, SIPRA Lab. Ltd., Trivedi Science, Trivedi Global, Inc., Trivedi Testimonials, and Trivedi Master Wellness for their assistance and support during this work

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Lupine Publishers | The Impact of Daily Kimchi Consumption: A Pilot Study

 Lupine Publishers | Scholarly Journal of Food and Nutrition

Abstract

Background: Kimchi, a traditional fermented Korean food, contains prebiotics and probiotics, which have demonstrated ability to impact irregular gastrointestinal (GI) symptoms.

Objective: To evaluate the impact of daily kimchi consumption on GI symptoms, sensory characteristics and consumer acceptability of kimchi, and the microbial content in homemade and commercial kimchi.

Design: Dietary intervention study incorporating an experimental design. Participants consumed 75g (½ cup) of kimchi twice a day for 14 days. Instruments included 3-day food records, a modified Gastrointestinal Symptom Rating Scale (GSRS), stool diaries using the Bristol Stool Scale (BSS), and a 7-point Hedonic scale (for sensory analysis and consumer acceptability). Lactic Acid Bacteria (LAB) were enumerated via plating methods.

Participants/Setting: Participants (n=20) experiencing irregular GI symptoms were recruited from a Mountain West university community during fall 2017.

Main Outcome Measures: Main outcomes included GI symptoms, tracking of bowel movements, pre and post response to consumer acceptability, and LAB levels in homemade and commercial kimchi.

Analysis: Analysis included: 1) repeated measures ANOVA (p< 0.05) for the GSRS and BSS with post-hoc testing for mean comparison between symptoms, 2) paired T-tests to assess mean differences in consumer acceptability and nutrient intake, and 3) culture-based microbial analysis with surface plating methods to determine bacterial counts.

Results: Participants reported a significant decrease in abdominal pain, heartburn, acid regurgitation, abdominal rumbling and distention, and eructation and gas production. Consumer acceptability and sensory characteristics questionnaires showed a majority of participants ‘liked’ kimchi and were willing to consume kimchi in the future. Bacterial analysis showed homemade kimchi had a lower concentration of LAB compared to commercial kimchi.

Conclusions: Kimchi is a nutrient dense probiotic source, acceptable among consumers in the study, with potential to impact irregular GI symptoms. Understanding consumer perception of kimchi provides valuable insight to when kimchi may be suggested as a method of probiotic and prebiotic intake.

Keywords:Kimchi; gastrointestinal; consumer acceptability; fermented foods; bacterial content; probiotics

Introduction

Fermentation has ancient roots in a variety of cultures. Kimchi, a fermented mixture of cabbage, salt, red peppers, radishes, and a variety of spices, has been a staple to Korean culture for over 4,000 years. [1,2] Lactic acid producing bacteria (LAB) produced during fermentation have been identified as factors that help maintain and improve gastrointestinal (GI) health. [3-5] Irregular GI symptoms affect 14.1% of the total U.S population. [6] The effects of irregular GI symptoms are associated with a severe decrease in quality of life, and a substantial financial burden on society. [6] The dysbiosis of the microbiome and resulting irregular GI symptoms may be induced by a wide variety of circumstances that include consumption of a Westernized diet which is typically high in fat and refined carbohydrates and low in dietary fibre. [7] Supplementation of probiotics may mitigate dysbiosis of the microbiome. [8] Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. [9] Research on the use of probiotics to alleviate dietary related GI disorders has shown potential. [3,4,10] With current research demonstrating positive effects with supplementation of LAB, [4,10] and the consumption of a variety of probiotics to maximize diversity and discourage the growth of harmful bacteria, [11] kimchi could provide a viable source of beneficial probiotics.

The bacteria promoted during kimchi fermentation have been demonstrated to act safely and effectively as probiotics. [12] Kimchi also contains prebiotics, such as inulin, a major food source for the growth of probiotic bacteria. Increased efficacy of probiotic supplementation has been demonstrated when prebiotics are present, lending credibility to claims that consumption of kimchi could positively affect the microbiome and improve irregular GI symptoms. [3,11] However, there is a paucity of literature addressing fermented foods in the Western diet and potential impact on the microbiome and GI symptoms. This pilot study examined 1) the impact of habitual consumption of kimchi on irregular GI symptoms and bowel form, 2) consumer acceptability and sensory characteristics of kimchi and 3) the microbial content in homemade and commercial kimchi.

Methods

During fall 2017, healthy participants (n=20) aged 18-40 years, experiencing irregular GI symptoms, but without history of malabsorptive or inflammatory bowel disease were recruited for a dietary intervention. Exclusion criteria included one or more of the following: diagnosed hypertension (due to high sodium content of kimchi), antibiotic use within past 3 months, current consumption of fermented products, excessive alcohol consumption (>3 drinks/day) (due to potential impact on GI microbiome) [13] and suppressed immune function (due to presence of live bacteria in kimchi). A sample size of 20 was estimated to provide an effect size of 0.8. [14] After initial phone screening to confirm eligibility, written, informed consent was obtained. The study was approved by the Institutional Review Board at the University of Wyoming. For this pilot intervention, participants consumed 75 grams (~½ cup) of kimchi twice a day for 14 days. Gastrointestinal symptoms were assessed at initiation, day 7, and day 14 using the Gastrointestinal Symptom Rating Scale (GSRS) [18] to evaluate the presence and severity of common symptoms associated with food intake.

The GSRS measures abdominal pain, heartburn, acid reflux, nausea and vomiting, borborygmus (stomach rumbling), abdominal distention, eructation (belching), gas production, decreased or increased passage of stools, hard or loose stools, urgent need to defecate, and satisfaction of bowel movement. The GSRS was modified from initial scale (0-3) to allow for consistency with other study questionnaires and each symptom was rated using a 7-point Likert Scale, where 1=no discomfort/presence of symptom and 7=severe discomfort/high frequency. Participants were asked to evaluate each bowel movement over the 14-days using the BSS (a common clinical tool used to evaluate GI transit time). [16] The BSS is a visual scale that depicts 7 types of common bowel forms, ranging from watery diarrhea with no solids (type 7) to constipation depicted as hard, separate lumps (type 1). Bowel formation of 1 or 2 was categorized as slow, 3 or 4 was normal, and 5-7 was fast.

The total number of slow, normal, and fast were represented as a percentage of total bowel movements during the week. Sensory characteristics and overall palatability were assessed by a 7-point Hedonic scale (7=extremely like, 1=extremely dislike) to rate appearance, flavour, texture, aroma, mouthfeel, and overall acceptability. Consumer acceptability and feasibility of including kimchi in their typical diet was assessed using a 7-point Likert scale (7=strongly agree, 1=strongly disagree) for five statements: ‘I enjoy consuming fermented foods as part of my regular diet’, ‘I am aware of different types of fermented foods and how to prepare them’, ‘I am interested in learning more [5] about the different types of fermented foods’, ‘I am willing to eat kimchi in the future’, and ‘I plan on consuming kimchi regularly’. [17] Participants recorded a three-day food diary (including two weekdays and one weekend day) during each week of the study. Training on tracking dietary intake, portion sizes, and guidance to continue with regular diet patterns was provided by the lead researcher and a registered dietitian prior to the initiation of the study.

Food records were examined at the exit interview with discrepancies in portion sizes or unclear entries clarified to increase accuracy. Diaries were utilized to compare and contrast oral dietary intake during the study period and to promote adherence to the kimchi intervention and avoidance of other fermented foods. Threeday food records were analyzed with dietary analysis software (ESHA Food Processor Nutrition Analysis Software, ESHA Products, Salem, OR 2018).

Bacterial Analysis

Culture-based microbial analysis of the fermented kimchi was conducted to enumerate LAB. Three major genera are known to predominate within traditionally fermented kimchi; Leuconostoc, Lactobacillus, and Weissella. [18] Bacterial enumeration was conducted using surface plating on de Man, Rogosa, and Sharpe (MRS) agar (Thermo Fischer Scientific). X-gal (5Bromo-4-Chloro- 3-Indolyl β-D-Galactopyranoside) additive was purchased through Millipore Sigma, previously Sigma-Aldrich, St. Louis, MO. Ten grams of kimchi from the commercial product and homemade kimchi (prepared by a culinary-trained researcher in a controlled campus teaching kitchen) were resuspended in 100ml of sterile saline (0.85% saline) solution in a WhirlPak® (Nasco, Fort Atkinson WI). The samples were mixed by hand for five minutes followed by serial dilutions using saline dilution blanks.

MRS agar or MRS agar supplemented with X-gal were utilized for surface plating. The supplementation with X-gal (chromogenic substrate for β6 galactosidase) provided additional differential discrimination for Leuconstoc spp.19 Leuconostoc spp. are known to synthesize β-galactosidase enzyme, thus colonies precipitate a blue color upon plating. MRS is commonly used to isolate and enumerate LAB.20Incubation was performed inside an anaerobic chamber containing anaerobic sachets (Thermo Scientific Oxoid anaerogen 2.5L Sachet) at 37˚ C for 72 hrs. Plating was performed in triplicates and counts were averaged to estimate kimchi microbial load as log10 CFU/g.

Statistical Analysis

Descriptive statistics were performed on all variables. Repeated measures ANOVA was used to assess significant changes in symptoms reported on the GSRS with post-hoc testing (Least Significant Difference, LSD) for mean comparison between symptoms. Frequency of slow, normal, and fast bowel movements reported on the BSS diaries were compared between the first and second week to identify improvement or changes in typical bowel formation and analyzed using repeated measures ANOVA. Posthoc testing using LSD determined differences between categories. Consumer acceptability pre- and post-intervention was analyzed using a paired T-test. Mean intake of key nutrients including macronutrients, dietary fibre, and sodium were compared from week 1 to week 2 of the study using paired T-test analysis. All statistics were performed using SPSS 24.0 (IBS SPSS Statistics for Windows, Version 24.0. Armonk, NY: IBM Corp 2016). The statistical significance was defined as p< 0.05 for all estimates.

Results

Forty individuals were screened with twenty-one individuals meeting inclusion criteria. One participant was lost to attrition on day 6 of the intervention. The GSRS and BSS were collected from individuals who completed the study. Consumer acceptability and sensory [7] characteristics included all twenty-one participants. Participant demographics are displayed in Table 1. Study compliance was assessed using food records or feedback at the exit interview. Gastrointestinal symptoms on the GSRS were grouped into six major categories (Table 2). Symptoms in categories 1, 3, and 4 were improved (p< 0.01) with kimchi consumption. Normal distribution was confirmed upon analysis using repeated measures ANOVA. Kimchi consumption had no measurable effect on typical stool form. The frequency of slow and normal bowel movements increased slightly, but not significantly (p=0.673).

Table 3 contains a complete breakdown of sensory characteristics. More than half (57.1%) of the study population ‘liked’ the kimchi, and an additional 9.5% “extremely liked” it. Aroma and appearance had the highest occurrence of ‘neither dislike nor like’ with 28.6% reporting for each. Minor changes in consumer acceptability were seen over the two-week period. The only statement to demonstrate a significant increase was ‘I enjoy consuming fermented foods as part of my regular diet’ (p=0.007). Comparison of average intake of calories, fat, protein, carbohydrates (CHO), dietary fibre, and sodium (Na) are presented in Table 4. Sodium data for one substantial outlier was excluded from the analysis. No significant differences between week 1 and 2 were detected for any of the selected nutrients.

Consumption of ½ cup kimchi twice a day and no additional fermented food intake was confirmed via the food diaries or verbally at the exit interview microbial counts of LAB were determined for both commercial and homemade kimchi. Commercial kimchi found LAB content when plated on MRS agar to be approximately 5.31 log CFU/g. Commercial kimchi plated on MRS with the addition of X-gal found content to be approximately 5.26 log CFU/g. Based on results obtained from plating on X-gal supplemented media, no detectable levels of Leuconostoc spp. were discernible. Homemade kimchi plated on MRS and MRS + X-gal, presented slightly lower concentrations of 4.21 log CFU/g and 4.26 log CFU/g respectively with no Leuconostoc spp. detected.

Discussions

An emerging number of clinical studies have evaluated the use of probiotics to improve GI disorder. The majority of these studies place emphasis on encapsulated probiotic supplements or probiotic enriched fermented milks/yogurts containing a mixture of LAB and Bifidobacterium spp. A number of these studies have demonstrated subjective improvement with regards to GI symptoms such as belching-abdominal fullness, bloating after meals, difficulty with defecation, and stomach gurgling. [3,21,22] Further research has demonstrated increased efficacy of probiotic supplementation when combined with prebiotic food sources such as inulin compared to probiotics alone. [3,23] Little research is available on the potential of fermented foods to act as beneficial probiotic sources.

Microbiological evaluation of kimchi has demonstrated the presence of a wide variety of LAB with the ability to act safely as probiotics. [12,24] However, limited information is [11] available for the use of kimchi to improve GI symptoms. Studies of probiotic supplementation generally emphasize high concentrations of bacteria and a wide variety of probiotic strains to maximize alleviation of symptoms. [11] In the current study, significant subjective improvement for abdominal pain, heart burn, acid regurgitation, abdominal rumbling and distention, and belching and gas production was observed, which is consistent with other studies. [3,21,22] Stool form was not significantly affected which was corroborated with non-significant changes on participants’ BSS. Previous studies have found that probiotic supplementation has little effect on typical stool forms. [4,22] Despite a recent rise in popularity of functional fermented foods, little is reported on consumer acceptability, sensory characteristics, and preference for kimchi. A previous study by Jang et al, evaluating the acceptability of kimchi presented similar findings to the current study in that there was an overall liking and perception of kimchi.25 Jang et al. found that in general, U.S. panelists gave overall liking scores of 6-7 on a 9-point hedonic scale.

The author described this as the liking of kimchi to be ‘slightly, to moderately pleasant.’ It should be noted that the higher liking of kimchi could have been related to participant’s preconceived notion about fermented foods and kimchi in general. It is plausible that some of the participants were attracted to the study due to the opportunity to incorporate kimchi into their regular diet. Food diaries provided insight about the typical diet of participants and allowed for comparison of key nutrients between each week. No significant changes in dietary intake of key nutrients was detected. Caloric intake is based on individual needs, so there are no set averages to assess participant caloric intake. According to the Institute of Medicine, Acceptable Macronutrient Distribution Range (AMDR), 20-35% of kcals should come from fat. [26] For the average 2278 kcal consumed by participants in this study, fat intake was 106 g which provided 41.8% of total kcal, [12] above AMDR recommendations.

The AMDR for protein is 10-35% of kcals. Participant protein intake was 90 g which provided 15.8% of total kcal, meeting the AMDR recommendation. Finally, the AMDR for CHO is 45-65% of kcals. Average CHO intake was 245g which provided 43.0% of kcal, just under the minimum recommendation. Diet, particularly dietary fibre, is a major component that influences GI function and microbiota. [27] Current recommendations for dietary fibre per day are 38g for men and 25g for women under age 50.26 Participant dietary fibre intake ranged from 13 g to 45 g with an average intake of 25g. The mean dietary fibre intake of all individuals 2 years and older in the U.S. population is 16g per day. [28] Kimchi contributed an additional 4g of dietary fibre per day per participant which contributed to dietary fibre intake higher than the national average.

Sodium consumption among participants was well above the 1,500 mg/day recommendation for both men and women, [29] with an average intake of 4,414mg. It was also above the average daily sodium intake (3,400 mg) for individuals over 2 years of age. [30] Consumption of kimchi contributed to this high average intake, as it added 1,400 mg of sodium per day. Kimchi has been evaluated for the impact an individual’s health. [24,31- 33] However, limited literature elucidates the potential for kimchi to act as a safe and effective probiotic source. [12,24,34] In the current study, bacterial evaluation denoted that the commercial kimchi had LAB concentrations of approximately 5. [31] log CFU/g, which is lower than the suggested concentrations found within traditional fermented kimchi (8log CFU/g) [35] and what is described on the commercial product packing (8.16 log CFU/g). Bacterial concentrations in kimchi are influenced by a variety of factors, specifically variations in raw materials such as vegetable type, harvesting area, season, and supplemental ingredients. [18] Furthermore, LAB has been described as comprising 68.7-98.1% of total bacteria present in kimchi. [35] This study was also not able to detect presence of 13 Leuconostoc spp. which are major contributors to the fermentation process.

The reasoning behind the lack of Leuconostoc spp. remains unclear. Leuconostoc spp. have been seen to predominate in the earlier stages of fermentation,18,36 thus microbial succession could be a reason. However, some strains such as, Le. gasicomitatum, have been seen to increase in later stages of fermentation, [37] which should be detectable by the plating methods utilized in our study due to their β-galactosidase activity.38 Despite the unclear reasoning for the lack of detectable Leuconostoc spp., this may prove to be advantageous for classifying kimchi as a probiotic rich food. Leuconostoc spp. have demonstrated low survival rates when passing through the GI tract31 which is vital for the classification as a probiotic. However, Lactococcus and Lactobacillus spp. have been validated to act as efficient probiotic bacteria. [24] While the microbial analysis followed previous literature for developing laboratory protocols [18,19] the study was limited to analysing presence of LAB, not total microbial content, thus estimations cannot be extrapolated to all commercial products. More so, while this study demonstrated improved symptoms, participants were aware of the intent of the study which could have induced the placebo effect.

Conclusions & Recommendations

Kimchi allows for inclusion of a nutrient dense vegetable source that has potential to impact GI health. Importantly, kimchi may provide a feasible and palatable method of supplementing probiotics within the diet and provides health professionals with an additional option to recommend to patients or clients. Further exploration of the impact of kimchi consumption would be beneficial in corroborating the evidence found within this study

 https://lupinepublishers.com/food-and-nutri-journal/fulltext/the-impact-of-daily-kimchi-consumption-a-pilot-study.ID.000120.php

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Lupine Publishers | Development and Application of HPLC/Post-Column Method for Analysis of Low Molecular Weight Organic Acids in Beers, Wines and Fruit Juices

 Lupine Publishers | Scholarly Journal of Food and Nutrition

Abstract

A post-column reaction method, which involves use of the pH indicator BTB, was developed and applied to the analysis of low molecular weight organic acids in commercial beers, wines, and fruit juices. Organic acids identified in these beverages were formic, acetic, citric, pyruvic, tartaric, malic, succinic, lactic, and pyroglutamic. Amounts of total acids in samples ranged from 980.4 mg/L to 513.5 mg/L in beers, from 7,502.3 mg/L to 5,573.3 mg/L in wines, and from 11,162.8 mg/L (orange) to 2,995.8 mg/L (mango) in fruit juices. Citric acid was found in the greatest amount-ranging from 286.7 ± 4.5 mg mg/L to 139.0 ± 5.3 mg mg/L in beers. The greatest level of malic acid was in a wine with 4,248.4 mg/L. The method developed is applicable to determine amounts of organic acids, formic and acetic acids, in beverages quickly and accurately.

Keywords: Organic acids analysis; HPLC/post-column; Beers; Fruit juices; Wines

Introduction

Low molecular weight organic acids (LMWOAs), including formic, acetic, malic, citric, tartaric, lactic, succinic, and oxalic acid, are found in various beverages, such as beers, wines and fruit juices (citrus, apple, grape, and melon), as well as in some foods [1,2]. These acids contribute characteristic tastes to beverages. For example, formic acid possesses pungent odor and sour taste in proper dilution. It is particularly adaptable to the pineapple flavor. Acetic acid also has a pungent and stinging sour odor, but it gives a clean-sour and acid taste at dilute concentrations (lower than 1%) in water and been used in flavor compositions, such as butter, chocolate, grape strawberry and wine. Malic acid is present in plum, peach, apricot and related fruits and used for imitation fruit flavors, such as maple [3]. Citric acid is the major acid component of citrus species and possesses a clean acid taste in an aqueous solution [3,4]. In addition to the role of organic acids in taste and flavor of beverages, they also play an important role in quality of beverages, including beers [5], wines [6] and fruit juices [7]. A capillary electrophoresis (CE) with spectrophotometric method achieved successfully analysis of a limited number of LMWOAs [8].

This method has been widely used for LMWOAs analysis in foods and beverages [9]. However, the resolution and detection of LMWOAs formic and acetic, achieved better by chromatographic methods than by the spectrophotometric method [10]. In the case of the most commonly used gas chromatography (GC), a tedious derivatization of organic acids is required to prepare samples for analysis because they are highly soluble in water [11]. On the other hand, HPLC, which can take aqueous samples directly, has been widely used to analyze LMWOAs in water samples; such as wine, beer [12], vinegar and fruit juices [13], coffee [14] and alcoholic beverage meads [15]. A HPLC interfaced to enzyme reactor analyzed oxalic acid in fruit and vegetable juices [16]. After the electronspray ionization system was advanced to interface between HPLC and mass spectrometers (LC/MS), LC/MS application for the analysis of water-soluble chemicals, including LMWOAs, developed significantly [17]. However, separation of LMWOAs by HPLC, formic acid and acetic acid, remains relatively difficult.

In the present study, a previously reported simple and accurate post-column reaction method for LMWOAs [18] was improved and successfully applied to analyze LMWOAs, including formic and acetic acids, in beers, wines, and fruit juices.

Materials and Methods

Beverage Samples

All commercial beverages-10 kinds of beers, 6 types of wines (5 red and 1 white), and 25 kinds of fruit juices-were bought from a local market in Seoul, Korea.

Chemicals and Reagents

Bromothymol blue (BTB, 95%), sodium phosphate dibasic anhydrous (98.5%), sodium hydroxide (98%), perchloric acid (67– 72%), oxalic acid (99.5–100.2%), phosphoric acid (85%), citric acid (99.5%), tartaric acid (99.5%), malic acid (95–100%), quinin acid (100%), succinic acid (99%), fumaric acid (99%), lactic acid (85%), formic acid (95%), acetic acid (99.7%), pyroglutamic acid (99%) were purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO, USA) or TCI Chemical Co. Ltd. (Tokyo, Japan). The purified water was prepared with a Zener Power II (Human Co., Korea). A stock solution of standard organic acids (10g/L) was prepared in purified water for preparation of standard solutions and spike analysis.

Preparation of Beer and Wine Samples

Beer and wine samples (2 mL each) were filtered with an Arcadis Syringe Filter with 0.45 mm PVDF membrane (Waters Co, Milford, MA).

Preparation of Fruit Juice Samples

Well-mixed juice samples were centrifuged at 10,000 rpm for 5min at 4 ºC to collect the supernatant. The supernatant was filtered with an Arcadis Syringe Filter with 0.45mm PVDF membrane (Waters Co, Milford, MA) and were analyzed with HPLC.

Analysis of Organic Acids in Beverages

Figure 1 shows the systematic diagram of the post-column method used in the present study. A prepared beverage sample was injected (10mL) into an Agilent 1100 model HPLC equipped with a 5cm x 8.0 mm i.d. Shorex RS pack KC-LG guard column, which was connected to two 30 cm x 8.0 mm i.d. KC-811 separation columns in series. The mobile phase was a 3mmol perchloric acid solution with a 0.7 mL/min flow rate. Temperature of the separation columns was 80ºC. The eluate from HPLC was mixed with a reaction solution containing 0.2mmol bromothymol blue (BTB), 15 mmol Na₂HPO₄, and 2 mmol NaOH. The reaction solution was purged at a 0.7mL/ min flow rate in a T-way connector as shown in Figure 1. Adducts formed from the acids and BTB in a 50 cm x 0.25 mm i.d. stainless reaction coil was monitored with a UV detector at l = 440 nm.

 

Results and Discussion

As mentioned above, analysis of LMWOAs is a difficult process. There have been advances in HPLC/MS methods recently but some problems, such as insufficient resolution of formic acid and acetic acid, remain. In order to resolve these problems, we developed a post-column reaction method, which involved use of pH indicator BTB. This method and related theory were originally advanced nearly three decades ago [18]. Figure 2 shows an HPLC postcolumn chromatogram of standard acids. This chromatogram shows satisfactory resolution of low molecular weight acids for quantitation. Phosphoric acid peak in this chromatogram may come from a phosphate in a reaction solution. However, phosphoric acid is not organic acid, which is out of scope of this study. The limits of detection (LOD) of acids were 25.0mg/L for citric, malic and quinin, lactic; 12.5mg/L for formic and acetic; and 50mg/L for pyroglutamic. The limits of quantitation (LOQ) of each acid were 75.0mg/L for citric, malic, quinin, and lactic; 30.0 mg/L for formic; 50.0 mg/L for acetic; and 150 mg/L for pyroglutamic.

Organic Acids Found in Beer Samples

Table 1 shows the results of LMWOAs analysis in beer samples. There are three monoprotic acids (lactic, acetic and pyroglutamic), two diprotic acids (malic and succinic), and one triprotic acid (citric acid). Amounts of total acids in beer samples varied considerably, ranging from 1,801.3 mg/L (brand E) to 776.0 mg/L (brand D). Among the acids identified in the beer samples, citric acid was generally found in the greatest amount-ranging from 286.7 ± 4.5 mg/L (brand E) to 139.0 ± 5.3 mg/L (brand G), followed by lacticranging from 274.3 ± 5.6 mg/L (brand E) to 19.3 ± 2.2 mg/L (brand G), and pyroglutamic acid-ranging from 264.9 ± 1.4 mg/L (brand E) to 105.5 ± 2.4 mg/L (brand D. ) Previously, the electrophoresis method was successfully used to analyze LMWOAs in beers. Klampft [19] found citric acid with level of 193 ± 2.4 mg/L in Chinese rice beer, 171 ± 1.5 mg/L in white beer and 178 ± 1.1 mg/L in Lager beer. Cordeiro-Ramirez et al. [20] reported citric acid (ND – 59 ± 2 mg/L), fumaric acid (ND – 5.9 ± 0.7 mg/L), malic acid (106 ± 12 mg/L), pyroglutamic acid (92 ± 6 mg/L) in 6 different beers. Citric acid exhibited synergistic effects with lactic acid-producing bacteria toward inhibition of pathogenic bacteria grow [21]. The amounts found in these acids in the present study were like these reports. For example, the present study found citric acid with amounts ranging from 146.2 ± 3.8 mg/L brand D to 286.7 ± 4.5 mg/L in brand E. In addition, the present study found pyroglutamic acid with levels ranging from 105.5 ± 2.4 mg/L in brand D to 264.9 ± 1.7 mg/L in brand E. The level of acetic acid ranged from 98.2 ± 3.0 mg/L (brand C) to not detected (brand H) in the present study, whereas acetic acid was not reported from the studies conducted with the electrophoresis method.

Organic Acids Found in Wine Samples

Table 2 shows the results of LMWOAs analysis in wine samples. The total amounts of acids were similar in all wine samples, ranging from 7,052.3mg/L (brand B) to 5,573.3mg/L (brand C). Tartaric acid was generally found in the highest concentration, ranging from 2,599.9 ± 7.5mg/L (brand F) to 1,495.1 ± 5.6mg/L (brand B). Amounts of citric acid, malic acid and lactic acid varied significantly among brands. Lactic acid was found at high levels in brands A (2,497.1 ± 2.8mg/L), C (2,271.1 ± 5.7 mg/L), and E (2,954.6 ± 4.2mg/L), but at relatively low levels in brands B (169.8 ± 1.9mg/L), D (170.4 ± 6.3 mg/L), and F (101.5 ± 4.9mg/L). On the other hand, high levels of malic acid were found in brands B (4,248.4 ± 13.8 mg/L), D (2,403.2 ± 8.0mg/L), and F (2,248.8 ± 5.3mg/L), whereas its concentrations in A was only 35.6 ± 3.8 mg/L in, C was 17.3 ± 1.4 mg/L and in E was 58.7 ± 4.2mg/L. A similar trend was observed in the case of citric acid. A previous study found lactic and tartaric acids in Ribeiro Sacra wines as the predominant organic acid components [9]. Lactic acid and tartaric acid levels ranged from 3,784mg/L to 452mg/L and 1978mg/L 866mg/L. These results are consistent to the ones from the present study. As mentioned above, the quality of wines depends on, in part, acidity associated with the composition of acids [22]. The results in the present study demonstrate that the composition of acids in different brands of wines varies. Therefore, the analytical method developed can be useful in evaluating wine quality as well as a guide to improvement in the winemaking process.

Organic Acids Found in Fruit Juice Samples

Table 3 shows the results of LMWOAs analysis in fruit juice samples. Formic acid and acetic acid, which are the lowest and 2^nd lowest molecular weight acids among LMWOAs and difficult to determine by a CE method [10], were satisfactorily analyzed in the present study. The levels of formic and acetic acids found ranged from 19.8 ± 1.0mg/L (mulberry) to 4.0 ± 0.2mg/L (mandarin) and from 27.7 ± 2.7mg/L (tomato) to 17.7 ± 2.6mg/L (grape), respectively. Orange juice samples had the greatest total concentration of acids, ranging from 11,162.8 mg/L (brand D) to 8,469.5 mg/L (brand C), followed by mandarin juice samples, ranging from 10,683.1mg/L (brand C) to 5,560.6 mg/L (brand B) and grape juice samples, ranging from 5,208.2 mg/L (brand E) to 4,016.2 mg/L (brand C). Citric acid and malic acid were generally detected in higher levels than other acids, ranging from 10,270.1 ± 2.9mg/L (mandarin) to 36.9 ± 12.0mg/L (mulberry) and from 3,990.3 ± 11.6mg/L (Grape) to 66.9 ± mg/L (mango), respectively. The pear juice sample contained the lowest level of total acid with 596.2 mg/L. The total amounts of acids in mixed juices ranged from 7,237.2mg/L (brand F) to 4,105.8 mg/L (brand A), which are comparable to those of grape juices.

One previous report demonstrated satisfactory analysis of organic acids in fruit juices (apple, peach, pear and apricot) using HPLC with an ion- exclusion column [23]. Another previous study reported the amounts of citric, malic, quinin and tartaric acids in apple, orange, cranberry, white/red grapes, and pomegrante juices analyzed using LC/MS/MS [24]. Effect of malic acid for the inactivation of common food pathogens on fresh-cut lettuce was reported, suggesting that malic acid possesses some biological activity [25]. The presence of formic and acetic acids was, however, not reported in these studies. On the other hand, the present study found 9.5 ± 3.6 mg/L formic acid in pear juice, 4.0 ± 0.2 – 10.0 ± 0.8 mg/L in mandarin juice, 12.5 ± 0.5mg/L – 15.2 ± 2.4mg/L in orange juice, 13.4 ± 0.3 mg/L in grape fruit, 5.8 ± 0.5 – 10.7 ± 0.3 mg/L in grape juice, 6.6 ± 1.5mg/L in mango juice, 10.1 ± 0.7 mg/L in tomato juice, and 19.8 ± 1.0 mg/L in mulberry juice. In addition, acetic acid was determined in pear juice (8.4 ± 1.3 mg/L), grape juice (17.7 ± 2.6 mg/L – 21.1 ± 1.0 mg/L), mango (23.2 ± 0.5 mg/L), and tomato (27.7 ± 2.7 mg/L). However, it is difficult to compare the results from the present study to those from the previous study because the compositions of the juices vary in the different brands.

Conclusion

The present study demonstrates that the method developed was useful to determine LMWOAs, formic acid and acetic acid, present in samples with complex matrices of beverages. Acids composition are important to evaluate quality of beverages. Therefore, the method developed in the present study would useful to analyze LMWOAs levels for evaluating quality of beverages. The CE method is also well-established method for LMWOAs analysis. Therefore, it is recommended to use both methods, the present method for formic acid and acetic acid

 https://lupinepublishers.com/food-and-nutri-journal/pdf/SJFN.MS.ID.000119.pdf

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