First part of implementation
The first part of EggWhiteBrain will produce a drink based on egg white enriched with vitamin B-12 (Cyanocobalamin) and folic acid in industrial conditions. Egg white is chosen because it is a drink rich in high nutritional value protein, low in fat and cholesterol. Folic acid is available in various forms, either as folic acid or in the form of salts ([6S] -5-methyltetrahydrofolic acid, 5-formyltetrahydrofolate, as well as salts with calcium or glucosamine). There is evidence that the methylated form and / or glucosamine complex have greater bioavailability in the human body. The aim of the first part of the project will be the enrichment of egg white drink with 1mg B-12 and 1mg folic acid (methylated form).
These concentrations were selected based on clinical studies that have shown that these doses help improve cognitive function in patients with Mild Mental Disorder, as well as prevent Alzheimer’s disease. Dietary supplements also help restore low levels of B12 and folic acid and treat high homocysteine ​​levels. In addition, this dosage complies with the regulations of the legislation both in terms of safety and in terms of consumption limits (SUP limits).

The following studies will be done for the production of the drink:
1. Solubility of B-12 and folic acid in egg white, in order to determine the maximum amount of vitamins that can be dissolved in the egg white (μg of vitamin / ml of drink) and their effect on the rheological and organoleptic characteristics of the drink.
2. How to introduce the drink (simple stirring, homogenization at high speed or pressure). The vitamins will be added to the egg white in different ways and will be stored under 4oC storage conditions. At regular intervals, samples will be taken from different parts of the container to determine the concentration of vitamins, while at the same time you will monitor for the formation of suspended particles or precipitation.
3. Determination of the amount of vitamins that are destroyed during the heat treatment (pasteurization) of the drink. The determination of the concentration of vitamins will be done using LC-MSMS technique.
4. Study of stability of vitamins in the drink. Samples of fortified beverages will be stored in real storage conditions (storage, 4oC) throughout the life of the product (~ 4 weeks) and their concentration in the beverage will be determined at regular intervals without prior agitation to determine whether precipitation of vitamins.
5. Based on the above results, the protocol of integration (initial quantities and method of introduction) of vitamins in the drink will be determined, so that the final product contains the desired quantities of vitamins with the least possible alteration of its original quality characteristics.
Second part of implementation
Two groups of patients with Mild Mental Disorder diagnosed according to the Morris 2012 criteria will be examined, a total of 30 people and a group of healthy volunteers of 15 people of the same age and gender. Patients will be informed in detail about the aims of the study and will give written consent to participate in it.
Patients will be randomized into two groups, who will receive the product as follows:

a) the first group one daily and
b) the second group one day after another

for a period of 12 months .

Neuropsychometric testing will be performed with the Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MOCA) tests before receiving the product after 6 months and after 12 months. During the same time period, the levels of folic acid, vitamin B12, and homocysteine ​​will also be measured.

Neuroimaging – Neurophysiological indicators

Neuroimaging with MRI of the brain and determination of the hippocampal tumor which is the main imaging indicator of Mild Mental Disorder and Alzheimer’s disease, as well as neurophysiological evaluation of cognitive function by performing cognitive provocative potential after receiving the product and after 12 months.
Third part of implementation
To better determine the beneficial effect of the dietary supplement on health, a thorough study of biochemical changes that take place at various levels of biological organization will be performed. For this purpose, a combined study of transcription and metabolism will be performed between volunteers who will consume the preparation and in a control group, which will not consume it. Blood and urine samples will need to be taken for analysis.
A combination of chromatography and mass spectrometry methods (GC / MS-MS / QToF Agilent 7200A, LC / MS-MS / QToF Agilent 1290-6540 UHD, LC / MS-MS Thermo Orbitrap) will be used to fully cover the synthesis of the metabolite as well as nuclear magnetic resonance spectroscopy (NMR 600 MHz Agilent). The combined use of these different technologies has been found to be able to give us the widest range of metabolites of different properties, where in turn they can give us more information about the subsequent analysis of metabolic pathways. Transcript analysis will be performed on a selected number of samples (50).
Transcript analysis will be performed with the Agilent SureScan using the SurePrint G3 Human Geb Exp v3 Array kit. The results of the analysis will be evaluated using the quantitative real-time polymerase chain reaction (RT-PCR). The results of transcript and metabolic analyzes will be analyzed in combination with bioinformatics tools. The analysis will be implemented in R language and in the Bioconductor standards, a methodology for finding “important” biomarkers in a biological network, which will include the entry of gene and metabolic expressions (txt. Or .raw files), quality control using Main Component Analysis (PCA) method, noise reduction due to errors in sample preparation and analysis, normalization and finding of differentiated biomarker expressions based on analysis of variance (ANOVA).
The results of the group analyzes from the population study will be mapped to regulatory pathways, performing common path analysis using GeneSpring software. The aim is to integrate the results from the group analyzes into a mechanistic description of the interactions of the pathways that affect the synthesis of vitamin B12 and folic acid, but also the beneficial effects of these dietary supplements with biomarkers related to better health, with emphasis on child neurodevelopment during pregnancy.
Fourth part of implementation
The aim of the 4th part will be production of 3 different products σκόνης αυγού από a) egg white, b) yolk, c) compination of a and b
The production of powder either from egg white or from yolk will be done using spray dryer. The process will consist of the following steps:

Initial processing (egg washing) → separation (separation of egg white from yolk, homogenization) → pasteurization (45 ° C, 30 min) → natural fermentation to remove sugars → spray drying → cooling (−40 ° C for 10 h) → sterilization (microwave drying oven) → cooling and sieving (standard sieve 80 mesh).

The following drying conditions will be specifically studied following a Box – Behnken statistical multifactorial design:

a) Supply inlet temperature

b) Concentration of supply

c) Power input speed

d) Drying air speed and temperature
The optimal conditions in a spray dryer will be determined in order to maximize the solid performance of the dryer, with the least possible degradation of the quality characteristics (color, -SH content, hygroscopicity and swelling ability). In addition, different microorganisms and enzymes for fermentation will be studied (glucose oxidase, LAB bacteria, etc.) to determine the optimal final quality of the product (taste, color and stability).
Finally, there will be research for the full economic and commercial utilization of the project results, which will include, among others, the Product Life Cycle Analysis, the planning of the strategy for the successful placement and acceptance of new products by consumers and its techno-economic evaluation. company AVGODIATROFIKI after the production of new products.
Expected results
  • Development of a new range of products for the company, with the creation of a functional food, but also egg powder.

  • Production of new knowledge regarding the metabolic pathways affected by the intake of fortified beverage.

  • Generating new knowledge about whether vitamin B-12 and folic acid in the form of supplements incorporated into a drink help prevent MMD.

  • Improving the competitiveness of the company by adding new products, opening to new markets (nutritional supplements) and increasing human labor, as the creation of new specialized human resources will be required.

  • Mechanistic description of the interactions of the pathways that affect the synthesis of vitamin B12 and folic acid, but also the beneficial effects of these dietary supplements with biomarkers related to better health, with emphasis on child neurodevelopment during pregnancy.
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