WO2022186710A1 - The method of obtaining orodispersible powder with immunoglobulins y and lysozyme - Google Patents
The method of obtaining orodispersible powder with immunoglobulins y and lysozyme Download PDFInfo
- Publication number
- WO2022186710A1 WO2022186710A1 PCT/RO2022/000002 RO2022000002W WO2022186710A1 WO 2022186710 A1 WO2022186710 A1 WO 2022186710A1 RO 2022000002 W RO2022000002 W RO 2022000002W WO 2022186710 A1 WO2022186710 A1 WO 2022186710A1
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- ylop
- lysozyme
- obtaining
- immunoglobulins
- powder
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/57—Birds; Materials from birds, e.g. eggs, feathers, egg white, egg yolk or endothelium corneum gigeriae galli
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
- A61K9/0056—Mouth soluble or dispersible forms; Suckable, eatable, chewable coherent forms; Forms rapidly disintegrating in the mouth; Lozenges; Lollipops; Bite capsules; Baked products; Baits or other oral forms for animals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/145—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
Definitions
- the use of one or more of the food preservation processes involves various additional technological operations. As a result of the products undergo physical, chemical and even biochemical changes. Usually, after processing, the taste and nutritional value are improved.
- preservation processes are based on the following biological principles: biosis, anabiosis, cenoanabiosis and abiosis. Food is protected through preservation against a number of external factors and must be carried out both during processing and during storage. The following factors are of interest:
- Food packaging must protect the product and act as a barrier, stopping or reducing the exposure to light, temperature or other physical agents that could lead to deterioration of the qualitative properties of the products (40, 44).
- the role of the barrier also refers to the gas transfer from the inside to the outside.
- Biological protection aims to maintain the hygienic and microbiological quality of food.
- food packaging must prevent both the entry of micro-organisms from the atmosphere or which may be taken from contact with certain surfaces, persons, etc., and the creation of favorable conditions for the appearance and development of pathogens (43).
- Modified atmosphere packaging and vacuum packaging are mainly used for products sensitive to the action of oxygen (e.g., meat and meat products, high fat products), which could be degraded even by very small amounts of oxygen (left in the package) during a longer period of storage. Both methods have the same purpose (elimination of oxygen), the choice of one or the other is conditioned by the characteristics of the product to be packaged (39).
- the modified atmosphere packaging consists in extracting oxygen from the inside of the package and replacing it with inert gases such as nitrogen and carbon dioxide.
- Nitrogen (N2) is inert, odorless and slightly soluble in water and fats; the purpose of its use is to reduce the oxidation of fats.
- Vacuum packaging consists in reducing the air pressure inside the package by means of a special installation. It is indicated for products sensitive to friction, ensuring their integrity and contraindicated for pressure sensitive products, which can be damaged or destroyed.
- Inert gases are generally used to avoid unwanted chemical reactions that degrade a product. These unwanted chemical reactions are often oxidation and hydrolysis reactions, favored by oxygen and moisture in the air (41).
- inert gases Due to the non-reactive properties of inert gases, they are often used to prevent unwanted chemical reactions. Food is packaged in inert gas to remove oxygen. This prevents the growth of bacteria and prevents the chemical oxidation of susceptible substances, such as fats. In food packaging, inert gases are used as a passive preservative, as opposed to active preservatives such as sodium benzoate (an antimicrobial) or BHT (an antioxidant).
- Oxidation of fats takes place in the range of 20 - 140 ° C and leads to the formation of aldehydes, ketones, free or oxidized fatty acids, triglycerides dimerized by oxygen bridges, etc. Oxidation is accelerated, in addition to temperature, by the air contact surface.
- Oxidation can be stopped by processing in the atmosphere of inert gases (nitrogen, argon, etc.) and thus the oxygen needed for the oxidation reaction is replaced by bioinhibiting agents, through a process of anoxianabiosis.
- inert gases nitrogen, argon, etc.
- Poultry eggs have long been recognized as an excellent source of nutrients and biological factors with immunomodulatory, antibacterial, antiviral, antifungal action (immunoglobulins, ovotransferins, lysozyme, ovomucin, ovomacroglobulin, etc.) (12, 15, 22).
- immunomodulatory, antibacterial, antiviral, antifungal action immunomodulatory, antibacterial, antiviral, antifungal action
- the biological factors with immunomodulatory, antibacterial, antiviral, antifungal action (immunoglobulins, ovotransferins, lysozyme, ovomucin, ovomacroglobulin) which are found in the orodispersible powder with immunoglobulins Y and lysozyme have a probiotic effect in the digestive tract. (6-9).
- Immunoglobulins are avian antibodies secreted naturally in the egg, mostly quantitatively and they have similar functions like the mammalian G immunoglobulins which are present in colostrum. Immunoglobulins Y intervene in the secondary immune response having a role in neutralizing toxins, viruses, bacteria, bacterial opsonization and intensification of phagocytosis, antibody-dependent cell-mediated cytotoxicity (antibody-coated cells are destroyed by K cells) (12, 13, 23, 32).
- Lysozyme is an enzyme which can be found in plants and animals that plays an important role in the natural defense mechanism. It has the ability to damage the cell walls of Gram-positive bacteria by hydrolysis of peptidoglycans (14, 36). It can also kill Gram-negative bacteria if they damage the outer membrane before exposure.
- the therapeutic efficacy of lysozyme present in the egg is in fact based on its ability to control the growth of susceptible bacteria and to modulate the host's immunity against infections and decreased immune responses (24, 33-35).
- the first feature is based on evidence of the biological activity of this enzyme, the second feature has been discovered recently and it is of extreme importance because of the possibilities offered in regulating the functioning of the host immune system (28, 29).
- Antibiotic activity and immune-boosting effects are used together in the treatment of gastrointestinal infections, including those caused by therapeutic treatments (14, 24).
- the most promising data today refers to the prevention of bacterial karyogenesis and the treatment of cancer patients to improve the efficacy of anticancer drugs or allow the host to recover from immunosuppression caused by anticancer treatments (26, 37).
- GALT Gut Associated Lymphoid Tissue
- probiotics beneficial bacterial populations in the gut known as probiotics, with functions in digesting food, absorbing nutrients, generating energy and releasing hormones.
- probiotic bacterial flora develops and functions at its optimum potential and thus ensures a normal immune function.
- Normal digestive function leads to a normal immune response.
- Orodispersible, fast-disintegrating and disintegrating forms of medicine are a modern form of drug administration. They were relatively recently introduced in therapy and they are an alternative to conventional oral preparations (solutions, suspensions, tablets or capsules).
- the excipients used in the formulation are closely related to the manufacturing technology and the characteristics that must be given to these powders.
- disintegrants or super- disintegrants are important categories in the dissolution and disintegration of orodispersible forms.
- a suitable disintegrant with an optimal concentration ensures rapid disintegration and high dissolution rates.
- Carbohydrates represent another category of excipients which are used to mask the taste and also for filling.
- the most used carbohydrates are mannitol, sorbitol, xylitol, dextrose, fructose. They are soluble and they give a pleasant sensation in the mouth and a good masking of the taste. Carbohydrates also give crystallinity, texture and elegance to the products.
- numerous studies demonstrate the preservative action of various carbohydrates on mammalian and avian immunoglobulins (1 - 4).
- orodispersible formulas are obtained by applying several manufacturing technologies like: lyophilization (freeze-drying or cryodrying), sublimation, spray-drying, casting (compression or heating), mass extrusion and direct compression.
- Freeze-drying is a process that was originally used for drying thermolabile substances, it was also successfully used as a method of preparing new pharmaceutical forms.
- the high porosity the most important characteristic of lyophilized products, has led to the use of products as fast-disintegrating pharmaceutical forms.
- many studies on lyophilization have focused on the identification and testing of new excipients that can provide sufficient mechanical strength, higher drying rate, increased stability overtime and a rapid dissolution of the active ingredient (10 , 11).
- the object of the present invention represent the method of obtaining and characterizing orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function by applying at least two combined processes of anabiosis (dehydration, osmoanabiosis, anoxianabiosis, cryoanabiosis etc.) with superior palatability - orodispersibility, intended for nutritional support, immune and probiotic, antimicrobial and immunomodulatory function.
- anabiosis dehydration, osmoanabiosis, anoxianabiosis, cryoanabiosis etc.
- Partial dehydration is a drying process based on reducing the water content, respectively increasing the concentration of dry matter, to values that allow the stability of the food to be preserved.
- the enzymatic activity is slowed down until stagnation and the development of microorganisms is stopped.
- the powder obtained through processing in the absence of oxygen is stable from microbiological, physical and chemical aspect.
- Packaging in a controlled / vacuum atmosphere and in a multilayer barrier film protects and maintains the product obtained for longer periods.
- the excipients used can be various, they are natural substances (carbohydrates / carbohydrates / sugars / salt / fiber / fructooligosaccharides, etc.) that are known to be the most effective and safe preservatives.
- the main advantage of natural preservatives is that, unlike the artificial ones, well-known E's, they do not cause health problems.
- a research program was carried out with the aim of obtaining an original, orodispersible, concentrated, preserved food / food supplement processed under controlled temperature and atmosphere conditions, with the addition of excipients and packaging in multilayer bags, optionally in vacuum or inert gas.
- the product can be stored at room temperature for a long time, being organoleptic, microbiological and physico-chemical stable.
- the obtained powders concentrate and preserve the nutritious, biologically and immunologically active elements contained in the raw egg.
- the products can be used as a food supplement, with nutritional, probiotic, antimicrobial and immunomodulatory functions.
- the low water content and the processing in inert gas ensure the stability of the products. This increases the shelf life of these products and they can be stored at temperatures below 25 ° C, maintaining their organoleptic, physico-chemical and microbiological characteristics.
- the products are obtained using natural ingredients concentrated through dehydration at moderate temperatures that keep the bilogical factors of interest bioactive (immunoglobulins, vitamins, lysozyme, etc.). Excipients are added to preserve and conserve the biological and nutraceutical properties of the active ingredients which are valuable for health.
- the method of production consists in dehydrating at a moderate temperature, in a controlled atmosphere, a liquid mixture made from eggs and / or active ingredients with indications for health, excipients and flavors by freeze-drying or other drying methods. Laboratory techniques and clinical trials were used to characterize the Y.L.O.P.:
- Y.L.O.P. brings a supply of valuable nutrients, complete, easily assimilated, natural, in a concentrated form. It contains proteins and fats with essential structural elements like amino acids and fatty acids, immunoglobulins, vitamins, minerals and enzymes.
- the product has a high energy value and can be consumed people of all ages.
- the only contraindications and restrictions on consumption is related to allergies to any of the ingredients.
- Orodispersible powder with Y immunoglobulins and lysozyme - YLOP - with nutritional, probiotic, antimicrobial and immunomodulatory function may contain:
- immunomodulatory, antibacterial, antiviral, antifungal action immunomodulatory, antibacterial, antiviral, antifungal action (immunoglobulin, ovotransferins, lysozyme, ovomucin, ovomacroglobulin, etc.)
- the method of obtaining orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory functions consists in the use of dehydration techniques at moderate temperatures that protect / maintain the thermolabile structures of interest unaltered (immunoglobulins, enzymes, etc.) in the presence of support substances - excipients and controlled atmosphere. These processes preserve and stabilize the product, protect the bioactive components and ensure their bioavailability.
- the present invention refers to the production and characterization of orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function, for any combination of whole eggs, egg components or whole egg elements, optionally ingredients with health recommendations and the use of excipients and flavors, under controlled temperature and atmosphere conditions.
- the method according to the invention consists in processing in the form of orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function of eggs and optionally with ingredients with health recommendations, by dehydration / drying at temperatures of maximum 75 o C in an atmosphere of N 2, in the presence of excipients and flavors.
- the orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function can be obtained from:
- excipients starch, salt, sucrose, stevia sweetness, honey, molasses, agave syrup, maple syrup, maltodextrin, mannitol, starch, dextran, lactose, fiber, FOS, etc.
- other supporting substances starch, salt, sucrose, stevia sweetness, honey, molasses, agave syrup, maple syrup, maltodextrin, mannitol, starch, dextran, lactose, fiber, FOS, etc.
- the raw egg or its components are homogenized with a mixture obtained from excipients, ingredients with recommendations for health and flavor.
- the mixture is processed through dehydration (lyophilization, atomization, drying in a fluidized bed, etc.) in an atmosphere of inert gas - N2.
- the mixture thus obtained is subjected simultaneously to the action of at least two processes of anabiosis which result in the production of orodispersible powder.
- the first process is xeroanabiosis by partial dehydration, through the use of heat and / or cryodissecation.
- the second process is osmoanabiosis by the adding excipients and anoxianabiosis - atmosphere of inert gas N2 bioinhibitory agent.
- the obtained product is packed in containers made of material that act as a protective barrier for light, water vapor and gas. This prevents the rehydration of the powder.
- the product can be packed in vacuum or inert gas. The technological flow is shown in Figure 2.
- the palatability of the product is imporved and the raw taste of the egg fades.
- the solubility of the powder is improved and gives it orodispersible properties.
- the solubilized excipients in the mix recrystallize and they give the resulting product a porous but hard texture at the same time. This allows the production of a granular powder by sieving and / or grinding the lyophilisate.
- the controlled atmosphere reduces the action of oxygen on the microorganisms and fats in the product, stopping the growth of microorganisms and the oxidation / rancidity of the products, which gives microbiological and physico-chemical stability to the obtained powder.
- Orodispersible powder with immunoglobulin Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function is obtained from whole egg, or combinations of various egg components or immunologically active ingredients from egg / optional and other ingredients with health indications, in different proportions , which ensures a minimum content of active ingredient: immunoglobulins, vitamins, minerals, growth factors, transfer factors, enzymes, lysozyme, collagen fibers, etc. .
- the egg and the ingredients with health indications are homogenized with a mixture of excipients in the necessary proportion, so that by drying the content and activity of the water are reduced below the limit that ensures the microbiological stability of the product.
- the mixture is subjected to drying at optimum temperatures in the atmosphere of N2, up to a maximum water content which ensures the preservation of the powder, by methods which preserve the structure and biological activity of the components of interest in the raw egg and those added for health, lysozyme, ovotransferins, vitamins, minerals, enzymes, etc.).
- the powder is packed in multilayer bags, optionally in inert gas or vacuum.
- Y.L.O.P. is preserved and stabilized. It can be stored, transported and marketed as a food ingredient / food supplement, with nutritional, probiotic, antimicrobial and immunomodulatory function.
- the control M sample of lyophilized whole egg remained in the form of agglomeration in the oral cavity. It does not disperse and it is very difficult to solubilize.
- the unpleasant odor, taste and aroma of raw egg powder are present. Total palatability is low due to the unpleasant taste, smell and aroma sensation obtained from chewing and tasting the control powder obtained only from the egg.
- the diffusimetric method was used to test the lysozyme content.
- the diameter of the halo is proportional with the concentration of the lysozyme that lyses the germs included in the medium.
- the lysozyme concentration was calculated by interpreting the values on the standard curve using standard concentrations. Measures to ensure the quality of the results were the use of the lysozyme standard and the ATCC certified bacterial strain.
- oxidation fats are broken down into free fatty acids AGL, lactic acid and OH-butyric acid which can be determined quantitatively through biological methods.
- Maximum limits for the quality of these compounds in egg products are set in the food quality standards.
- the allowed lactic acid content in egg products is 1000mg / kg, and that of hydroxy butyric acid is 10mg / kg.
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Abstract
The invention relates to the food industry. To obtain an orododispersible powder, eggs are sorted and disinfected, separated into ingredients, auxiliary substances are added, and the mixture is homogenized. Then they are dehydrated in an N2 atmosphere using at least two suspended animation methods, then the resulting powder is packaged and packaged.
Description
THE METHOD OF OBTAINING ORODISPERSIBLE POWDER WITH IMMUNOGLOBULINS Y AND LYSOZYME
APLICATION DOMAIN:
Nutraceutical industry, food supplement, biotechnology, anabiosis, excipients, orodispersibility, probiotic, antimicrobial, immunomodulator
PRESENTATION OF THE TECHNICAL STAGE
Due to their chemical composition, food provide an environment which favor the development of microorganisms and it is an excellent source for energy procurement and metabolic activities.
Nutrients are subject to the action of biological, physical, chemical factors in the manufacturing process and in the sales chain. Many raw materials and foods deteriorate easily and their availability is considerably shortened (38, 42).
Preservation ensures increased food stability and prolonged shelf life. In this respect, the use of food stabilization procedures is used. (44)
The use of one or more of the food preservation processes involves various additional technological operations. As a result of the products undergo physical, chemical and even biochemical changes. Usually, after processing, the taste and nutritional value are improved.
The ultimate goal of preservation by using a particular process, or by combining multiple preservation processes is to inhibit the physico-chemical changes inside the products and to stop or slow down the multiplication of microorganisms that inhabit the products, so that the availability period of food storage gets imporeved as much as possible. Preservation processes are based on the following biological principles: biosis, anabiosis, cenoanabiosis and abiosis.
Food is protected through preservation against a number of external factors and must be carried out both during processing and during storage. The following factors are of interest:
- physical factors: the relative humidity of the air, dust particles in the atmosphere, foreign bodies, light, temperature, etc.
- chemical and physico-chemical factors: air, water, oxygen, C02etc.
- biological factors: microorganisms, insects, etc.
Food packaging must protect the product and act as a barrier, stopping or reducing the exposure to light, temperature or other physical agents that could lead to deterioration of the qualitative properties of the products (40, 44).
From a chemical and physico-chemical point of view, it is very important that the product does not come into contact with aggressive chemicals such as H2, NH3, S02, C02, through vapors or gases produced by volatile substances (hydrocarbons, smoke, perfumes, etc.).
In this case, the role of the barrier also refers to the gas transfer from the inside to the outside. In order to avoid the loss of the specific aromas of the product, its dehydration, the loss of the gases introduced in the packaging in order to preserve the products, etc. Biological protection aims to maintain the hygienic and microbiological quality of food. To that end, food packaging must prevent both the entry of micro-organisms from the atmosphere or which may be taken from contact with certain surfaces, persons, etc., and the creation of favorable conditions for the appearance and development of pathogens (43).
Modified atmosphere packaging and vacuum packaging are mainly used for products sensitive to the action of oxygen (e.g., meat and meat products, high fat products), which could be degraded even by very small amounts of oxygen (left in the package) during a longer period of storage. Both methods have the same purpose (elimination of oxygen), the choice of one or the other is conditioned by the characteristics of the product to be packaged (39).
The modified atmosphere packaging consists in extracting oxygen from the inside of the package and replacing it with inert gases such as nitrogen and carbon dioxide. Nitrogen
(N2) is inert, odorless and slightly soluble in water and fats; the purpose of its use is to reduce the oxidation of fats.
Vacuum packaging consists in reducing the air pressure inside the package by means of a special installation. It is indicated for products sensitive to friction, ensuring their integrity and contraindicated for pressure sensitive products, which can be damaged or destroyed.
Inert gases are generally used to avoid unwanted chemical reactions that degrade a product. These unwanted chemical reactions are often oxidation and hydrolysis reactions, favored by oxygen and moisture in the air (41).
Due to the non-reactive properties of inert gases, they are often used to prevent unwanted chemical reactions. Food is packaged in inert gas to remove oxygen. This prevents the growth of bacteria and prevents the chemical oxidation of susceptible substances, such as fats. In food packaging, inert gases are used as a passive preservative, as opposed to active preservatives such as sodium benzoate (an antimicrobial) or BHT (an antioxidant).
Oxidation of fats takes place in the range of 20 - 140 ° C and leads to the formation of aldehydes, ketones, free or oxidized fatty acids, triglycerides dimerized by oxygen bridges, etc. Oxidation is accelerated, in addition to temperature, by the air contact surface.
Dehydration processing of high fat mixtures in the presence of atmospheric oxygen can lead to their oxidation, rancidity and degradation of the end product.
Oxidation can be stopped by processing in the atmosphere of inert gases (nitrogen, argon, etc.) and thus the oxygen needed for the oxidation reaction is replaced by bioinhibiting agents, through a process of anoxianabiosis.
Poultry eggs have long been recognized as an excellent source of nutrients and biological factors with immunomodulatory, antibacterial, antiviral, antifungal action (immunoglobulins, ovotransferins, lysozyme, ovomucin, ovomacroglobulin, etc.) (12, 15, 22). In terms of the chemical composition of the egg, it is a complete food, containing proteins, fats, minerals, vitamins, enzymes valuable essential fatty acids (5, 27, 30, 31).
The immunologically and biologically active components of the egg can be stored through appropriate techniques into products that ensure their bioavailability and can be administered to humans and animals as immune, nutritional support, with multiple health indications.
The biological factors with immunomodulatory, antibacterial, antiviral, antifungal action (immunoglobulins, ovotransferins, lysozyme, ovomucin, ovomacroglobulin) which are found in the orodispersible powder with immunoglobulins Y and lysozyme have a probiotic effect in the digestive tract. (6-9).
Y immunoglobulins (IgY) - are avian antibodies secreted naturally in the egg, mostly quantitatively and they have similar functions like the mammalian G immunoglobulins which are present in colostrum. Immunoglobulins Y intervene in the secondary immune response having a role in neutralizing toxins, viruses, bacteria, bacterial opsonization and intensification of phagocytosis, antibody-dependent cell-mediated cytotoxicity (antibody-coated cells are destroyed by K cells) (12, 13, 23, 32).
Genetic research has classified avian IgY as the evolutionary precursors of mammalian immunoglobulins IgG and IgE.
By consuming the egg, the biological factors of interest in its composition are usually destroyed by the heat treatment applied. IgY in the egg are thermolabile and are destroyed by high temperatures. Vitamins and enzymes are also thermolabile (12, 25). Lysozyme is an enzyme which can be found in plants and animals that plays an important role in the natural defense mechanism. It has the ability to damage the cell walls of Gram-positive bacteria by hydrolysis of peptidoglycans (14, 36). It can also kill Gram-negative bacteria if they damage the outer membrane before exposure. The therapeutic efficacy of lysozyme present in the egg is in fact based on its ability to control the growth of susceptible bacteria and to modulate the host's immunity against infections and decreased immune responses (24, 33-35). While the first feature is based on evidence of the biological activity of this enzyme, the second feature has been discovered recently and it is of extreme importance because of the possibilities offered in regulating the functioning of the host immune system (28, 29). Antibiotic activity and immune-boosting effects are used together in the treatment of gastrointestinal infections, including those caused by therapeutic treatments (14, 24). Based on these
biological properties, in addition to the wide range of therapeutic activities for which lysozyme has been used in the past, the most promising data today refers to the prevention of bacterial karyogenesis and the treatment of cancer patients to improve the efficacy of anticancer drugs or allow the host to recover from immunosuppression caused by anticancer treatments (26, 37).
It is known that 70-80% of the immune system is located in the digestive tract (GALT - Gut Associated Lymphoid Tissue). The body develops beneficial bacterial populations in the gut known as probiotics, with functions in digesting food, absorbing nutrients, generating energy and releasing hormones.
When ingesting food we bring into the gastrointestinal tract microorganisms and toxic substances, which disrupt the normal digestive flora, compete with it and produce imbalances in the functioning of digestion, implicitly in the functioning of associated lymphoid formations. All the above produce an imbalance of the immune response. Biologically active factors in YLOP powders act on these disruptive invaders of the digestive flora, through various mechanisms of destruction and / or neutralization and they transmit signals to the immune system which in turn sends into battle all the elements available to balance the immune status (13, 23) .
In this way the beneficial, probiotic bacterial flora develops and functions at its optimum potential and thus ensures a normal immune function. Normal digestive function leads to a normal immune response.
Orodispersible, fast-disintegrating and disintegrating forms of medicine are a modern form of drug administration. They were relatively recently introduced in therapy and they are an alternative to conventional oral preparations (solutions, suspensions, tablets or capsules).
These medicinal forms have the distinctive feature of being able to disperse and dissolve rapidly in saliva once placed in the oral cavity, before being swallowed. Their dispersal, disintegration or dissolution takes place in a few minutes, without being chewed and without requiring water for administration.
Currently, the interest in these modern forms of administration is growing, mainly due to the advantages they have compared to other known medicinal forms, even compared to the usual tablets. Rapidly dissolving / disintegrating formulas are very suitable for
administration to patients who have difficulty swallowing (dysphagia), especially in the elderly and children (16, 17). These forms have several advantages:
- They represent an ideal pharmaceutical form for medicinal substances that are absorbed orally. They may be suitable forms for the oral release of therapeutic proteins or peptides, which have limited bioavailability when administered in conventional tablets, such as IgY (7-9).
- They mask the unpleasant taste of some medicinal substances, improving compliance, especially for children (20, 21). It masks the raw taste of the egg in the case of the products formulated in this patent.
- Compared to oral liquid products, they are much easier to transport and allow an accurate dosage when administered (18,19).
The excipients used in the formulation are closely related to the manufacturing technology and the characteristics that must be given to these powders.
An important category of excipients is represented by disintegrants or super- disintegrants. They play an important role in the dissolution and disintegration of orodispersible forms. A suitable disintegrant with an optimal concentration ensures rapid disintegration and high dissolution rates.
Carbohydrates represent another category of excipients which are used to mask the taste and also for filling. The most used carbohydrates are mannitol, sorbitol, xylitol, dextrose, fructose. They are soluble and they give a pleasant sensation in the mouth and a good masking of the taste. Carbohydrates also give crystallinity, texture and elegance to the products. In addition, numerous studies demonstrate the preservative action of various carbohydrates on mammalian and avian immunoglobulins (1 - 4). Currently, orodispersible formulas are obtained by applying several manufacturing technologies like: lyophilization (freeze-drying or cryodrying), sublimation, spray-drying, casting (compression or heating), mass extrusion and direct compression.
Freeze-drying is a process that was originally used for drying thermolabile substances, it was also successfully used as a method of preparing new pharmaceutical forms. The high porosity, the most important characteristic of lyophilized products, has led to the use of products as fast-disintegrating pharmaceutical forms. In recent years, many studies on lyophilization have focused on the identification and testing of new excipients
that can provide sufficient mechanical strength, higher drying rate, increased stability overtime and a rapid dissolution of the active ingredient (10 , 11).
When developing a system with rapid dissolution / disintegration, other objectives are taken into account, such as the need to mask the taste of the drug substance and the incorporation of the active substance into the product matrix.
Brief description of technical solutions
The object of the present invention represent the method of obtaining and characterizing orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function by applying at least two combined processes of anabiosis (dehydration, osmoanabiosis, anoxianabiosis, cryoanabiosis etc.) with superior palatability - orodispersibility, intended for nutritional support, immune and probiotic, antimicrobial and immunomodulatory function.
Partial dehydration is a drying process based on reducing the water content, respectively increasing the concentration of dry matter, to values that allow the stability of the food to be preserved. By reducing the humidity of the products, the enzymatic activity is slowed down until stagnation and the development of microorganisms is stopped. The powder obtained through processing in the absence of oxygen is stable from microbiological, physical and chemical aspect. Packaging in a controlled / vacuum atmosphere and in a multilayer barrier film protects and maintains the product obtained for longer periods.
The excipients used can be various, they are natural substances (carbohydrates / carbohydrates / sugars / salt / fiber / fructooligosaccharides, etc.) that are known to be the most effective and safe preservatives. The main advantage of natural preservatives is that, unlike the artificial ones, well-known E's, they do not cause health problems.
In order to establish the method for obtaining YLOP orodispersible powder, a research program was carried out with the aim of obtaining an original, orodispersible, concentrated, preserved food / food supplement processed under controlled temperature and atmosphere conditions, with the addition of excipients and packaging in multilayer bags, optionally in vacuum or inert gas. The product can be stored at room temperature for a long time, being organoleptic, microbiological and physico-chemical
stable. Thus, the obtained powders concentrate and preserve the nutritious, biologically and immunologically active elements contained in the raw egg. The products can be used as a food supplement, with nutritional, probiotic, antimicrobial and immunomodulatory functions.
The main advantages of the obtained products are:
The low water content and the processing in inert gas ensure the stability of the products. This increases the shelf life of these products and they can be stored at temperatures below 25 ° C, maintaining their organoleptic, physico-chemical and microbiological characteristics.
The products are obtained using natural ingredients concentrated through dehydration at moderate temperatures that keep the bilogical factors of interest bioactive (immunoglobulins, vitamins, lysozyme, etc.). Excipients are added to preserve and conserve the biological and nutraceutical properties of the active ingredients which are valuable for health.
Adding excipients and flavors increases palatability and has improved the solubility which gives the powder its orodispersible properties. This is important as IgY is also absorbed in the oral cavity. It also reduces the hygroscopic nature of the powders. They no longer absorb water vapor from the environment.
Various active ingredients of different origins (vegetable, mineral, animal, etc.) can be incorporated and the powder can be used to manufacture other forms of presentation (tablets, drops, etc.). This leads to obtaining products with multiple indications for health.
The method of production consists in dehydrating at a moderate temperature, in a controlled atmosphere, a liquid mixture made from eggs and / or active ingredients with indications for health, excipients and flavors by freeze-drying or other drying methods. Laboratory techniques and clinical trials were used to characterize the Y.L.O.P.:
- Lysozyme content by agar gel immunodiffusion technique
- Sensory testing and "in vivo" tests of the characteristics that print the orodispersibility of the products
- Clinical evaluation of probiotic function and digestive reaction following powder administration
- Paraclinical assessment of increased nonspecific resistance - by monitoring cellular effectors - leukocytes
- Fat content and the degree of degradation and oxidation
Y.L.O.P. brings a supply of valuable nutrients, complete, easily assimilated, natural, in a concentrated form. It contains proteins and fats with essential structural elements like amino acids and fatty acids, immunoglobulins, vitamins, minerals and enzymes.
The product has a high energy value and can be consumed people of all ages. The only contraindications and restrictions on consumption is related to allergies to any of the ingredients.
Orodispersible powder with Y immunoglobulins and lysozyme - YLOP - with nutritional, probiotic, antimicrobial and immunomodulatory function may contain:
- biological factors with immunomodulatory, antibacterial, antiviral, antifungal action (immunoglobulin, ovotransferins, lysozyme, ovomucin, ovomacroglobulin, etc.)
- substances with recognized nutritional and biological value (proteins, fats, carbohydrates, growth factors, amino acids and essential fatty acids, vitamins, minerals, enzymes, etc.).
- ingredients with health recommendations of various origins (vegetable, mineral, animal, etc.)
The method of obtaining orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory functions consists in the use of dehydration techniques at moderate temperatures that protect / maintain the thermolabile structures of interest unaltered (immunoglobulins, enzymes, etc.) in the presence of support substances - excipients and controlled atmosphere. These processes preserve and stabilize the product, protect the bioactive components and ensure their bioavailability.
The present invention refers to the production and characterization of orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function, for any combination of whole eggs, egg components
or whole egg elements, optionally ingredients with health recommendations and the use of excipients and flavors, under controlled temperature and atmosphere conditions.
DETAILED DESCRIPTION OF THE INVENTION
The method according to the invention consists in processing in the form of orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function of eggs and optionally with ingredients with health recommendations, by dehydration / drying at temperatures of maximum 75 o C in an atmosphere of N 2, in the presence of excipients and flavors.
The orodispersible powder with immunoglobulins Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function can be obtained from:
- whole egg, whole egg and addition of nutritious / biologically active components isolated from whole egg, egg white, egg yolk, membranes, shell, etc.
- any other combination of egg components or elements isolated from the whole egg.
To the eggs we add the following:
- optionally ingredients with health recommendations (of vegetable, mineral, animal origin)
- excipients (starch, salt, sucrose, stevia sweetness, honey, molasses, agave syrup, maple syrup, maltodextrin, mannitol, starch, dextran, lactose, fiber, FOS, etc.), other supporting substances.
- flavorings (vanilla, cocoa, strawberries, bananas, etc.).
The raw egg or its components are homogenized with a mixture obtained from excipients, ingredients with recommendations for health and flavor. The mixture is processed through dehydration (lyophilization, atomization, drying in a fluidized bed, etc.) in an atmosphere of inert gas - N2.
The mixture thus obtained is subjected simultaneously to the action of at least two processes of anabiosis which result in the production of orodispersible powder. The first process is xeroanabiosis by partial dehydration, through the use of heat and / or
cryodissecation. The second process is osmoanabiosis by the adding excipients and anoxianabiosis - atmosphere of inert gas N2 bioinhibitory agent.
The method of production leads to the following results:
- reduction of contact with oxygen during the drying process and elimination of the risk of multiplication of microorganisms and oxidation of fats in the product
- obtaining a powder with orodispersible characteristics, with a higher solubility and palatability than a powder made only from egg, with a reduced hygroscopic character
- obtaining a preserved powder by dehydration, addition of excipients, processing and packaging in a controlled atmosphere
- keeping, preservation and concentration of nutrients and bioactive components, and ensuring their bioavailability (fats, proteins, enzymes, IgY, lysozyme, vitamins, minerals, etc.)
- integration / incorporation of other active bilogical factors, besides those found in the egg, ingredients with health recommendations
The obtained product is packed in containers made of material that act as a protective barrier for light, water vapor and gas. This prevents the rehydration of the powder. Optionally the product can be packed in vacuum or inert gas. The technological flow is shown in Figure 2.
By adding excipients and flavors the palatability of the product is imporved and the raw taste of the egg fades. Also, the solubility of the powder is improved and gives it orodispersible properties. In the dehydration process, the solubilized excipients in the mix recrystallize and they give the resulting product a porous but hard texture at the same time. This allows the production of a granular powder by sieving and / or grinding the lyophilisate. The controlled atmosphere reduces the action of oxygen on the microorganisms and fats in the product, stopping the growth of microorganisms and the oxidation / rancidity of the products, which gives microbiological and physico-chemical stability to the obtained powder. Y.L.O.P. provides the body essential nutrients, biological factors, active ingredients and immunological components, in a concentrated, preserved and stabilized formula.
METHOD OF OBTAINING LYOSISME AND IMMUNOGLOBULINS Y ORRODISPERSIBLE POWDER WITH NUTRITIVE, PROBIOTIC, ANTIMICROBIAL AND IMMUNOMODULATORY FUNCTIONS
Orodispersible powder with immunoglobulin Y and lysozyme with nutritional, probiotic, antimicrobial and immunomodulatory function is obtained from whole egg, or combinations of various egg components or immunologically active ingredients from egg / optional and other ingredients with health indications, in different proportions , which ensures a minimum content of active ingredient: immunoglobulins, vitamins, minerals, growth factors, transfer factors, enzymes, lysozyme, collagen fibers, etc. .
The egg and the ingredients with health indications are homogenized with a mixture of excipients in the necessary proportion, so that by drying the content and activity of the water are reduced below the limit that ensures the microbiological stability of the product.
The mixture is subjected to drying at optimum temperatures in the atmosphere of N2, up to a maximum water content which ensures the preservation of the powder, by methods which preserve the structure and biological activity of the components of interest in the raw egg and those added for health, lysozyme, ovotransferins, vitamins, minerals, enzymes, etc.). The powder is packed in multilayer bags, optionally in inert gas or vacuum.
Y.L.O.P. is preserved and stabilized. It can be stored, transported and marketed as a food ingredient / food supplement, with nutritional, probiotic, antimicrobial and immunomodulatory function.
RECOMMENDED MODELS FOR OBTAINING AND CHARACTERIZING THE
INVENTION
The five models presented below are intended to illustrate and are not intended to limit the scope of the present invention.
Obtaining the orodispersible powder by dehydration at temperatures below 75 o C in N 2 atmosphere of the whole egg homogenate, with the addition of excipients and Vanilla Bourbon flavor.
1. “In vivo” evaluation of formulas obtained with various carbohydrates
The formulations obtained by lyophilization of whole egg in N2 atmosphere, with the addition of excipients and vanilla were evaluated "in vivo". The evaluation consisted of measuring the time of disintegration / solubilization in the oral cavity of a quantity of 3g of powder, the appreciation of the taste, the volume of the residue and the total palatability. (Table 1)
Table 1 - "In vivo" evaluation of various dehydrated fomulas
The control M sample of lyophilized whole egg remained in the form of agglomeration in the oral cavity. It does not disperse and it is very difficult to solubilize. The unpleasant odor, taste and aroma of raw egg powder are present. Total palatability is low due to the unpleasant taste, smell and aroma sensation obtained from chewing and tasting the control powder obtained only from the egg.
Test conclusions:
The variants processed with excipients are easily and uniformly dispersed in the oral cavity. This property was given by the addition of excipients, which achieves the granulation of the finished product and its disaggregation. At the same time, it imprints the solubilization of the product. This property is not found in the control sample with plain whole egg, without the addition of excipient. Solubilization is performed differently depending on the concentration in excipients. It takes between 3-5 minutes, being faster in the case of formulas which are more concentrated in excipients. The tests were repeated periodically every 6 months for 2 years. The results were similar to the initial ones.
2. Testing for lysozyme content
The diffusimetric method was used to test the lysozyme content.
The principle of the method: on a plate in which the culture of Micrococcus lysodeicticus strain ATCC is incorporated, the samples of the product to be analyzed are applied in gel wells. Diffusion of lysozyme From the samples forms a halo around the well (Figure 1).
The diameter of the halo is proportional with the concentration of the lysozyme that lyses the germs included in the medium.
The lysozyme concentration was calculated by interpreting the values on the standard curve using standard concentrations. Measures to ensure the quality of the results were the use of the lysozyme standard and the ATCC certified bacterial strain.
Four samples of powerder with different carbohydrates 12g powder per 100ml a.d. were agitated / dissolved / sedimented / centrifuged. Three dilutions were used (Table 2)
Table 2 - Lysosyme test results by diffusimetric method
Test conclusions:
In the tested variants of lyophilized powder of the finished product, it was observed that:
- lysozyme is found in all tested variants
- the excipients and flavorings used have mentained the activity of lysozyme
- the different concentrations of excipients did not affect the activity of lysozyme, which is found in all test formulations obtained through lyophilization
The results show that:
- the technology used preserves and maintains bioactivity of egg lysozyme in the powder
- the addition of excipients and flavorings does not affect the activity of lysozyme
- lysozyme has an antimicrobial effect on the reference strain Micrococcus lysodeicticus strain ATCC which demonstrates the antimicrobial activity of the lyophilised powder
3.Clinical and paraclinical findings on the probiotic, immunomodulatory and digestive effects of YLOP
3.1 Orodispersible powder with immunoglobulins Y and lysozyme was administered to elderly patients aged 67-92 years with multiple pathologies, under permanent medical observation, for 8 weeks (Table 3)
Table 3 Clinical and paraclinical findings on probiotic, immunomodulatory effect and digestive response in elderly patients
Conclusions:
- the digestive transit was improved and various digestive problems (bloating, flatulence, stool appearance, diarrhea, constipation, infections) were remedied. This demonstrates the probiotic effect
- resistance to diseases increased, main diagnostic symptoms were improved and the efficiency of the applied treatments increased
- improved tone, more energetic, more active
- immunomodulatory effect was demonstrated by changes in the immunograms
3.2 Orodispersible powder with immunoglobulins Y and lysozyme was administered to children aged 17 months to 9 years, under the constant observation of their parents, for 8 weeks - 11 patients (Table 4, 5, 6).
Table 4 - Group structure of children and diseases presented
Observations on the evolution of the present diseases: amelioration, disappearance, accentuation of symptoms
Table 5 - Evolution of present diseases
Observations on immune status increase in resistance to disease, seasonal exposure / virus / parasitic communities, reduction of disease episodes in intensity and frequency, increased effectiveness of treatments applied in various diseases
Table 6 - Clinical assessment of immune status
Conclusions:
- Increased resistance to disease was observed after 60 days of administration.
- There has been an increase in the effectiveness of treatments for respiratory viruses after 60 days.
- Symptoms have been reported to improve or disappear
4.Laboratory results on increasing nonspecific resistance - by monitoring cellular effectors - leukocytes - immunomodular function
Y.L.O.P. in patients with leukopenia diagnosed by paraclinical examinations - hemoleukograms, for two months after which the retest was performed. Patients underwent long-term treatments / infections that led to the onset of immunosuppression. The administration was performed in a non-hospital setting and the results were provided by the patients (Table 7).
Table 7 Results on the increase in the number of leukocites
Conclusions: for patients aged 25 to 70 years diagnosed with leukopenia, after the administration of the orodispersible powder with immunoglobulins Y and lysozyme, there was an increase in the number of leukocytes, cellular immunity effectors and obtaining the immunomodulatory effect.
5. Testing the content of AGL by enzymatic methods
Specific laboratory tests were performed using enzyme kits to verify the oxidation stage of YLOP fats. The powders have a high content of fats susceptible to oxidation in the presence of oxygen and at high temperatures.
By oxidation fats are broken down into free fatty acids AGL, lactic acid and OH-butyric acid which can be determined quantitatively through biological methods. Maximum limits for the quality of these compounds in egg products are set in the food quality standards. The allowed lactic acid content in egg products is 1000mg / kg, and that of hydroxy butyric acid is 10mg / kg.
Determinations performed on YLOP by enzymatic tests revealed very low amounts of lactic acid and hydroxybutyric acid, well below the limits set by food standards. Conclusions: the method of obtaining in a controlled atmosphere, in the absence of oxygen creates adequate processing conditions and protects the fats in the structure of
the product, they are not degraded by oxidation aspect demonstrated by laboratory results (Table 8).
Table 8 Results of enzymatic tests on the content of free fatty acids
Claims
1. The claim that the YLOP method is a method of producing powder of whole eggs or any combination of / and / from eggs, excipients and optionally added ingredients with health recommendations.
2. The claim that the method of obtaining YLOP is achieved by the simultaneous application of at least two processes of anabiosis (xeroanabiosis, osmoanabiosis, excipients addition , etc.).
3. The claim according to which in the method of obtaining YLOP the addition of excipient substances has the role of preservative, protective role, taste corrector, support mold, disintegrant, etc.
4. A claim that moderate temperatures and a controlled atmosphere are used in the method of obtaining YLOP that mantain active, concentrate and preserve the nutritional and biological components of interest.
5. The claim that the method of obtaining YLOP can be used as a method of incorporating various active ingredients with health recomandations.
6. The claim that by the applied method is obtained an orodispersible powder with multiple bioactive ingredients (immunoglobulins Y, vitamins, minerals, lysozyme, etc.) with improved palatability, disaggregation and solubility.
7. The claim that YLOP provides a minimum content of active ingredients: immunoglobulins, vitamins, minerals, growth factors, transfer factors, enzymes, lysozyme, collagen, etc. .
8. The claim that YLOP has probiotic, antimicrobial, immunomodulatory function.
9. The claim that YLOP can be used as a source of nutrients, macro and trace elements.
10. The claim that YLOP can be used to make other forms of presentation tablets, capsules, lollipops, drops, etc.
11. The claim according to which YLOP by the applied packaging method in vacuum or inert gases, in multilayer packaging with the role of protective barrier against external factors: gases, vapors, light, etc.
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