WO2023197052A1

WO2023197052A1 - Immunoglobulin-preserving method for producing powdered human milk, powdered human milk, kit containing powdered human milk, use of the kit and use of the powdered human milk

Info

Publication number: WO2023197052A1
Application number: PCT/BR2023/050103
Authority: WO
Inventors: Emily Fernanda Freire Folco; Maira BILLI
Original assignee: Eurofarma Laboratórios S.A.
Priority date: 2022-04-13
Filing date: 2023-03-28
Publication date: 2023-10-19
Also published as: BR102022007104A2

Abstract

The invention relates to an immunoglobulin-preserving method for producing lyophilised powdered human milk and to the kit for reconstituting the easily fractionated lyophilised material.

Description

“PROCESS FOR OBTAINING HUMAN MILK POWDER FOR PRESERVATION OF IMMUNOGLOBULINS, HUMAN MILK POWDER KIT CONTAINING HUMAN MILK POWDER, USE OF THE KIT AND USE OF HUMAN MILK POWDER”

Field of Invention

[0001] The present invention relates to the process of obtaining lyophilized powdered human milk for preserving immunoglobulins and to the kit for reconstituting the lyophilized material for easy fractionation. The present invention is in the fields of pharmacotechnics and pharmaceutical technology.

Fundamentals of Invention

[0002] Breastfeeding is the best way to nourish newborns up to 6 months of age, according to Health Organizations. Breastfeeding is understood as the act of feeding the baby with breast milk directly from the mother's breast.

[0003] During the lactation period, a period in which there is production of breast milk due to hormonal action, human milk is the first food option for newborns due to the nutritional profile and biological characteristics of milk. This is the most important and natural food, biologically classified as the gold standard in feeding newborns, especially for premature babies (babies born before 37 weeks of gestation) and extremely premature babies (babies who came into the world before 28 weeks of gestation). and their lives are at greater risk than babies born a few weeks later, as they are in a very fragile state of health).

[0004] Human milk is the lactic secretion produced by the nursing mother, where the nursing mother is the woman with lactic acid production (milk). This milk can have different classifications according to the lactation period. The first product of lactic secretion, obtained on average up to 7 days after birth, is called colostrum and has the main role of protecting the newborn and, for this reason, it is also known as the first vaccine. Follow-on milk is the product intermediate of the lactic secretion of the nursing mother, between colostrum and mature milk, obtained on average between the ^7th and ^15th day after birth. Finally, mature milk is the product of the nursing mother's lactic secretion, free from colostrum, obtained on average from the ^15th day after birth.

[0005] Breastfeeding has general positive effects on the health, growth and development of babies, and can often be used as a treatment for babies who need nutritional support.

[0006] It is increasingly becoming clear that the unique composition of human milk cannot be recreated or replaced by other types of milk, such as cow's milk or other animal milk, nor by artificial milk.

[0007] Nutritional components, bioactives and immunological factors from milk provide adequate defense against infections. This defense is modulated by immune responses and intestinal microbiota. Newborns have an immature immune system and need their mother's help, through breastfeeding, to protect themselves against infections. Therefore, feeding babies with other types of non-human milk makes the transmission of passive immunity impossible.

[0008] Passive immunity consists of the transfer of humoral immunity of ready and active antibodies, which occurs naturally through the placenta and human milk to the baby.

[0009] Among the immunoglobulins (lg) present in human milk, the one found in greatest quantity is the immunoglobulin IgA. This plays a crucial role in the baby's defense, being present on mucous surfaces against the entry of pathogenic bacteria and enteroviruses.

[0010] When breastfeeding is not possible, replacing breast milk with human milk, obtained through donation, is the best alternative. Human milk obtained by donation is understood as expressed human milk, which would be milk obtained through the milking procedure.

[0011] Human milk is well tolerated when compared to the administration of industrialized formulas. [0012] A diet consisting of human milk dramatically reduces the incidence of necrotizing enterocolitis, intolerances and can reduce the length of hospital stay, in addition to having greater nutritional quality.

[0013] Expressed human milk intended for consumption by newborns, particularly those admitted to Intensive Care Units (ICUs), must not contain microorganisms in quantity or quality capable of causing health problems. Therefore, it is necessary to have procedures capable of ensuring the sanitary quality of milk.

[0014] Pasteurization represents an effective alternative, long known and practiced in the field of food technology. Pasteurization is the heat treatment that expressed human milk must undergo to inactivate its microbiota.

[0015] As a standard procedure, the Global Network of Human Milk Banks recommends that raw expressed human milk collected and approved by quality control should be pasteurized at 62.5 °C for 30 minutes after the preheating time. Pasteurization does not aim to sterilize milk, but rather to guarantee the inactivation of 100% of potentially present pathogenic microorganisms, whether due to primary or secondary contamination, in addition to 99.99% of the saprophytic or normal microbiota.

[0016] Freezing is the most common method of preserving human milk during storage, however, the nutritional and immunological components are lost over time in this storage stage.

[0017] Faced with this scenario, freeze-drying shows promise as a preservation method, improving processes in Milk Banks. Freeze drying is understood as the process of removing water by sublimation and desorption.

[0018] The freeze-drying process is widely known and widespread around the world. Aiming to remove water from a material to extend stability, since chemical degradations can be caused by hydrolysis or be catalyzed in the presence of water. Therefore, with reduced humidity, it is possible to slow down the speed of these degradation reactions.

[0019] Freeze drying is composed of multiple steps, in which each step is critical and must be well controlled for the success of the process. Therefore, the scenario must be well known and the steps strictly controlled to obtain satisfactory results.

[0020] The advantages of this process lie in the rapid stabilization of the material, long-term stability, which results in a porous material that is easy to reconstitute and is a process that maintains the essential and original characteristics of the material.

[0021] Freeze drying consists of the process of freezing a material to form a solid, followed by drying steps, which aim to remove the frozen water or solvent from the material through sublimation and the water bound by the desorption process , without it returning to its initial, liquid state.

[0022] This technique can increase the storage time of human milk and minimize losses of nutrients, including proteins and triglycerides, immunological content and antioxidant integrity throughout storage and transportation time. In addition, the stability of some nutrients (vitamins, fatty acids and antioxidants, for example) is greater when milk is freeze-dried compared to simple freezing.

[0023] The great challenge in the state of the art is to develop a process for obtaining powdered human milk with IgA, IgM and IgG immunoglobulin retention values greater than 84%.

Brief Description of Figures

[0024] Figure 1 shows the comparison of the concentration of IgA, IgM and IgG immunoglobulins in mg/mL (axis on the left) for samples subjected to different freeze-drying cycles and percentage of retention of immunoglobulins (axis on the right) compared to the control sample.

[0025] Figure 2 shows the comparison of the concentration of IgA, IgM and IgG immunoglobulins in mg/mL for samples subjected to different freezing methods: P1 UF = ultrafreezer, P1 N = liquid nitrogen and P1 F = freezer.

[0026] Figure 3 shows the comparison of the concentration of total lg immunoglobulins (IgA + IgM + IgG) in mg/mL for samples subjected to different freezing methods: P1 UF = ultrafreezer, P1 N = liquid nitrogen and P1 F = freezer .

[0027] Figure 4 shows the amount of total protein in samples subjected to a single freeze-drying cycle, using different freezing methods: P1 UF = ultrafreezer, P1 N = liquid nitrogen and P1 F = freezer, where P1 = control.

[0028] Figure 5 shows the comparison of the concentration of IgA, IgM and IgG in mg/mL and retention percentage for frozen milk and freeze-dried milk samples.

[0029] Figure 6 shows the graph of the freeze-drying process according to parameters defined in the invention.

[0030] Figure 7 shows the reconstitution time values found for the freeze-dried colostrum and mature milk sample, the pH values found for the colostrum and mature frozen milk sample and the same milks after the freeze-dried process and the moisture values found for freeze-dried colostrum and mature milk samples.

[0031] Figure 8 shows the results found in the microbial count and presence of Bile Tolerant Gram-negative Bacteria for the mature milk sample before and after freeze-drying.

[0032] Figure 9 shows a freeze-dried human milk kit consisting of an aluminum sachet with a parabola bottom, a bottle with a lid containing 30mL of drinking water, 10mL syringes and an adapter and cartridge for packaging the components. Summary of the Invention

[0033] The present invention relates to the process of obtaining lyophilized human milk powder for preserving immunoglobulins and to the kit for reconstituting the lyophilized material that can be easily fractionated, as well as its use. Said process has the advantage of a degree of retention of IgA, IgG and IgM immunoglobulins in human milk greater than or equal to 84% in relation to frozen human milk.

[0034] The present invention presents the following objects as inventive concepts.

[0035] The present invention presents as its first object the process of obtaining human milk powder for preserving immunoglobulins, comprising the steps of human milk fractionation, freezing, drying and sealing.

[0036] The present invention presents as a second object human milk powder, obtained by the process defined in the first object and its modalities, comprising a degree of retention of IgA, IgG and IgM immunoglobulins greater than or equal to 84% in relation to milk frozen human.

[0037] The present invention presents as a third object a kit comprising an aluminum sachet with a parabola bottom containing lyophilized human milk powder as defined in the second object and obtained from the process defined in the first object and in its embodiments, a bottle with a lid containing drinking water, a syringe adapter, a set of disposable syringes and a cartridge for packaging all materials.

[0038] The present invention presents as a fourth object the use of the kit comprising the steps of opening the kit and arranging the components for use, pre-heating the bottle containing drinking water in a water bath at 40 °C, transferring the entire contents of the sachet into the bottle of pre-heated water, closing the bottle and homogenizing until complete reconstitution of the lyophilized human milk powder, checking that all the contents was solubilized, opening the bottle and positioning the syringe adapter and fractionating the milk into syringes according to the volume necessary for individual administration for immediate use of the reconstituted and fractionated milk, as described in the third object and its embodiments.

[0039] The present invention presents as a fifth object the use of human milk powder for the production of reconstituted human milk and a kit containing human milk powder, useful for the manufacture of a food formula that protects against infections, diarrhea and allergies and promotes healthy growth, weight gain and possible reduction in hospitalization time for premature babies.

Definition

[0040] In the present invention, the term “degree of immunoglobulin retention” or “percentage of immunoglobulin retention” refers to the relative percentage of viable immunoglobulin content after a certain process, considering the initial content. In the present invention, the degree of immunoglobulin retention is calculated considering: (i) lyophilization as a process; (ii) the initial immunoglobulin content before freeze-drying, i.e., the immunoglobulin content in frozen human milk; and (iii) the final immunoglobulin content after freeze-drying, that is, the immunoglobulin content in the human milk powder.

Detailed Description of the Invention

[0041] The present invention relates to a process for obtaining lyophilized powdered human milk for preserving immunoglobulins and to the kit for reconstituting the lyophilized material for easy fractionation. Said process was developed in an unexpected and surprising way, since there is no process in the state of the art with the advantage of having a degree of retention of IgA, IgG and IgM immunoglobulins from human milk greater than or equal to 84% in relation to milk frozen human. Additionally, the present invention also relates to the easily fractionated lyophilized human milk reconstitution kit for administration of up to 6 doses.

[0042] Initially, the components of the milk and, as it is a complex biological material, the main focus of the invention was immunological elements. In this way, some components were selected as quality markers and monitored during the milk freeze-drying development process, capable of significantly preserving immunoglobulins.

[0043] The main challenges for the mapping and significant preservation of immunoglobulins are: determining the best parameters to be used in the freezing and lyophilization process of human milk, capable of significantly preserving immunoglobulins, the development of a method capable of quantifying with acceptable accuracy the milk immunoglobulins and the correct choice of packaging material for adequate packaging of the freeze-dried milk.

[0044] Different freezing methods and freeze-drying parameters have been described to obtain freeze-dried human milk, with the preferred freezing process being deep-freezing and freeze-drying with vacuum pressure below 200pbar.

[0045] To carry out the process proposed in the invention, the step of defrosting the pasteurized milk may or may not be necessary, depending on the state of the material to be fractionated.

[0046] Thawing frozen human milk aims to transfer heat to frozen expressed human milk, in sufficient quantity to change the solid phase to the liquid phase, not allowing the final temperature of the product to exceed 5 °C. The defrosting time for human milk varies according to the volume, type of packaging and equipment used in the process.

[0047] The defrosting step must be carried out at 40 °C in a water bath with a water level above the milk level to allow adequate heat penetration. Defrosting should not be carried out at room temperature, as, in this condition, the surface of the milk thaws faster than its interior, and can reach temperatures that allow microorganisms to multiply. Defrosting must occur with the container in which the milk frozen is stored completely closed.

[0048] During defrosting, it is recommended to homogenize the milk with circular movements of the containers, preferably every 5 minutes, to guarantee its temperature distribution. When the milk is in liquid form, place the containers in an ice bath and send them to the next stage of the process.

[0049] The fractionation of human milk, coming from any nursing mother and at any stage of lactation, must occur in a controlled environment to avoid possible contamination. Fractionation must be carried out in a clean environment with standard procedures for cleaning, disinfection and sterilization of equipment, articles, materials and well-defined surfaces, and can be carried out in an environment with microbiological control, and/or preferably under laminar flow.

[0050] The fractionation step must occur with previously pasteurized milk, and can be carried out with the aid of a graduated, volumetric, automatic pipette, scale or other measuring instruments for standardization and precision of the filling volume.

[0051] The fractionation of human milk can occur in sachets, reagent bottles, ampoule bottles, syringes, aluminum cells, trays or other supports to contain the product on the freeze dryer shelves. In the present invention, human milk is divided into sachets, which are preferably made of aluminum and specifically in aluminum sachets with a parabola bottom.

[0052] The aluminum sachets are composed of polypropylene, polyethylene and other additives, free from Bisphenol A, also known as BPA. BPA can contaminate food, which can pose some health risks, such as hormonal changes and even embryo malformations, in the case of pregnant women. Studies on this component and its risks are still discussed. As a precaution, some countries, including Brazil, have chosen to ban the import and manufacture of baby bottles containing Bisphenol A, considering the greater exposure and susceptibility of individuals using this product. For this reason, packaging for processing and storing freeze-dried human milk must be free of this component.

[0053] The aluminum sachet maintains the smell, flavor and original color of the material, and is also an important protective barrier against oxidation, humidity and light. This material is chemically inert and stable, being resistant to low and high temperatures, that is, the material performs well and, for example, does not become brittle at low temperatures.

[0054] Aluminum sachets are easy to transport compared to other packaging due to their shape, size and weight. Furthermore, in the lyophilization process, during drying, most of the water in the material is removed, further reducing the final weight of the packaged material, in the case of the present invention, lyophilized human milk powder.

[0055] The proposed sachet must be used in the single dose version, with regard to the use of its entire contents when opened. Therefore, once opened, the product cannot be stored.

[0056] The proposed packaging avoids product leftovers in the corners and the determined packaging volume is ideal for consuming the entire contents, avoiding waste.

[0057] The aluminum sachet used for the human milk freeze-drying process has a rounded or parabola bottom, which prevents product loss when compared to straight bottoms with corners, where product accumulation may occur at the lower ends, specifically at the junction of the wall with the bottom of the sachet, forming a 90° angle, making it difficult to access and remove residual content. In this way, all the packaged material is consumed by the final recipient without any loss in packaging or waste. Furthermore, the packaging can be rinsed with drinking water present in the kit of the present invention to ensure the use of the entire contents of the packaging. [0058] As previously stated, the milk must be kept cold throughout the batch fractionation process. The volume to be divided must correspond to volumes compatible with a single dose, which guarantee that all the contents are used. This volume can vary from 5 to 30ml_. For volumes greater than 5ml_, fractionation of the milk is considered after reconstitution in individual syringes containing the necessary volume to be administered to the baby.

[0059] In the fractionation stage, the aluminum sachets are filled with the desired volume and positioned vertically in containers/supports that allow the sachets to be kept in a vertical position without risk of leakage. The aluminum sachets remain completely open at this stage.

[0060] After filling and positioning the sachets in the containers/supports, said sachets are protected by a plastic membrane to prevent moisture from entering and protect their contents. The freezing stage must take place at low temperatures, below -30 °C, preferably between -80 °C and -30 °C. The freezing time may vary according to the volume to be frozen, material and format of the packaging used, number of samples inside the container/support and temperature used during the process.

[0061] The freezing time can vary from 30 minutes to 5 hours depending on the filling volume of 5 - 30ml_, considering the equipment's temperature set point of -70 °C.

[0062] In terms of frozen material, low temperatures result in a decrease in aqueous activity, as 80% of the free water in the product is transformed into ice. Therefore, biochemical reactions and enzymatic activities in the product, as well as the action of microorganisms, are delayed or even inhibited.

[0063] After the freezing step, the sachets are positioned horizontally on the previously cooled shelves of the freeze dryer, which may or may not be aligned or overlapped, as long as the upper part of the sachet remains open and unobstructed for sublimation and desorption of the ice. If desired, the sachets can be positioned vertically to maximize the capacity of the freeze dryer, however the drying plateau time will be extended due to the smaller surface area available for heat exchange by conduction and/or exchange by direct contact with the shelf.

[0064] At the end of the drying cycle, the sachets are analyzed for the residual moisture of the freeze-dried milk and, if approved, they are sealed to hermetically close the packaging to avoid moisture gain from the freeze-dried milk. Preferably, sealing can be carried out under vacuum to remove oxygen from the packaging and inhibit oxidation processes.

[0065] Additionally, a kit was developed comprising a copy of said aluminum sachets with a parabola bottom containing lyophilized human milk powder, a bottle with a lid containing drinking water, a syringe adapter, a set of disposable syringes and a cartridge for packaging. of all materials in order to facilitate the administration of reconstituted human milk.

[0066] Said kit contains all the necessary materials for preparing and administering freeze-dried human milk, with the method of preparing freeze-dried human milk being characterized by opening the freeze-dried human milk kit, leaving all components of the kit available for use. Next, it is necessary to wash your hands and sanitize the area where the milk will be prepared, to avoid possible external contamination. The drinking water that comes with the kit must be previously heated in a water bath at 40 °C with the bottle closed. The water must be heated in its original packaging, a glass bottle with a lid. With the water heated, you can open the aluminum sachet containing the freeze-dried milk and pour the entire contents of the sachet into the bottle containing the previously heated drinking water. After pouring out all the content of the sachet, you can also rinse the packaging with the water provided in the kit to fully utilize the contents. The bottle must be closed completely and homogenized carefully until the milk is completely solubilized. The freeze-dried human milk according to the invention is quickly reconstituted and in less than a minute it is possible to perform a visual check to make sure that the entire content of the milk has been solubilized. With the milk already prepared, open the bottle and position the syringe adapter to divide the milk into individual portions, as needed. After opening the sachet and preparing the milk, it must be used immediately.

[0067] In view of the above, in an unexpected and surprising way, the present invention reveals the optimization of experimental freezing and drying conditions in order to develop a process for obtaining powdered human milk for preserving immunoglobulins, with the advantage of having a degree of retention of IgA, IgM and IgG immunoglobulins in freeze-dried human milk greater than or equal to 84% in relation to frozen human milk. In this context, the present invention is defined by the following objects and their embodiments.

[0068] In a first object, the process of obtaining human milk powder for preserving immunoglobulins comprises the steps of:

(a) fractionation of human milk;

(b) freezing;

(c) drying; It is

(d) sealing, in which the degree of retention of IgA, IgM and IgG immunoglobulins in freeze-dried human milk is greater than or equal to 84% in relation to frozen human milk.

[0069] According to an embodiment of the present invention, step (a) of fractionation comprises aliquoting human milk and filling it into sachets. [0070] According to an embodiment of the present invention, said sachets are made of aluminum and free from Bisphenol A.

[0071] According to an embodiment of the present invention, said sachets have a parabola bottom. This bottom prevents loss of content accumulated in the corners of the packaging.

[0072] According to an embodiment of the present invention, step (b) of freezing human milk occurs in a temperature range of -80 °C to -30 °C, in an interval of 30 minutes to 5 hours.

[0073] According to an embodiment of the present invention, freezing step (b) comprises:

(i) position the aluminum sachets containing human milk vertically in the freezing equipment, with or without the aid of containers and support supports;

(ii) protect the top of the opened sachets with a plastic membrane.

[0074] According to an embodiment of the present invention, step (c) of drying occurs in a freeze dryer, in which the open sachets containing frozen milk are positioned on the shelves of said freeze dryer in a horizontal or vertical position, in a row or overlapping manner. ; primary drying occurs at 20°C and 150pbar; and secondary drying occurs at 35°C and 150pbar; and, wherein the residual moisture of the freeze-dried human milk powder is less than or equal to 5.0%.

[0075] According to an embodiment of the present invention, in step (d) the welding time of said sealing of aluminum sachets is 2 to 8 seconds, with welding power of 1 - 5; and wherein said welding is preferably vacuum.

[0076] In a second object, powdered human milk, obtained by the process defined in the first object and its embodiments, comprises a degree of retention of IgA, IgG and IgM immunoglobulins greater than or equal to 84% in relation to frozen human milk . [0077] In a third object, the kit containing human milk powder, as defined in the first object and its embodiments, comprises an aluminum sachet with a parabola bottom containing lyophilized human milk powder, a bottle with a lid containing drinking water, a syringe adapter, a set of disposable syringes and a cartridge for packaging all materials.

[0078] In a fourth object, the use of the kit, as defined in the third object, comprises the steps of:

(a) open the kit and arrange the components for use;

(b) preheat the flask containing drinking water in a water bath at 40 °C;

(c) transfer the contents of the sachet to the bottle of preheated water;

(d) close the bottle and homogenize until complete reconstitution of the lyophilized powdered human milk;

(e) check whether all content has been resolved;

(f) open the vial and position the syringe adapter;

(g) divide the milk into syringes with the volume necessary for individual and immediate administration of the reconstituted and fractionated milk.

[0079] In a fifth object, the present invention discloses the use of powdered human milk, as defined in the first object and its embodiments, for the manufacture of a food formula that protects against infections, diarrhea and allergies and promotes healthy growth , weight gain and possible reduction in hospitalization time for premature babies.

Examples

[0080] The following examples serve to illustrate aspects of the present invention without, however, having any limiting character.

EXAMPLE 1 - Human milk preparation and freeze-drying process.

[0081] Freeze-dried human milk powder was prepared according to the procedure described below. [0082] 20ml of mature human milk were filled into transparent borosilicate glass bottles with screws. These bottles were placed in the Freezer of the duplex refrigerator (refrigerator/freezer) Sanyo model MPR-414F with a working range of -30 to -20 °C for 5 hours.

[0083] The freeze dryer shelves were prepared for loading the material at -15 °C and starting the freeze drying cycle.

[0084] The control sample, frozen mature human milk, was stored in the freezer until the date of sample analysis.

[0085] The sublimation and desorption of water from the sample was explored using different drying parameters, summarized in Table 1.

[0086] The milk after lyophilization was reconstituted with water for injection preheated to 40 °C, returning to the initial concentration of solids in the sample, and analyzed by the sandwich ELISA method to quantify immunoglobulins.

[0087] To quantify these samples, total milk protein analysis was carried out, following the Pierce® BCA Protein assay Kit protocol from the company ThermoScientific, following the manufacturer's instructions.

[0088] The measurements were taken on the UV-VIS spectrophotometer. The calibration curve was made using bovine serum albumin [2mg/mL] 95% purity, diluted in the same buffer as the sample, with readings taken at 570nm. In a 96-well plate, 25pL of samples are deposited and then mixed with 200pL of the mixture of reagents A + B (1/50 parts) as described by the manufacturer. The plate is incubated at 37 °C for 30 minutes and, after incubation, the absorbance measurements are read. After quantification of total milk proteins, the samples are subjected to analysis by sandwich ELISA and the results obtained are normalized by the amount of total protein, previously verified.

[0089] To carry out the sandwich ELISA method, the anti-lgA, IgM, IgG antibodies were diluted in phosphate buffer (PBS) pH 7.5 at dilutions of 1:5,000 (anti-lgA and anti-lgM), and 1 :1,000 (anti-lgG) in 96-well plates (NUNC Maxabsorb) and kept at 4 °C overnight for immobilization of capture antibodies. After incubation, they were washed and dried at 34 °C in an incubator for later use.

[0090] Powdered human milk samples were weighed to 15 mg each and diluted in 1 ml of ultrapure water at 40 °C. These samples were used for protein dosing and, after dosing, corrective dilution was made to deposit 1.5mg/ml per well of total protein (1%) of the weighed mass. These samples after dilution were incubated with the control samples IgA, (500-50pg) IgG (25-0.5pg) and IgM in dilutions 1 -100pg and incubated for 30 minutes at 37 °C in an oven.

[0091] After incubation, the plate was washed 5 times with PBS-T (0.5%) and again incubated with the secondary antibody at a dilution of 1:10,000 anti-lgA, (1:5000) IgG, (1:5,000) IgM conjugated with peroxidase for 30 minutes. After incubation, the plate was washed again with wash buffer (PBS-T 0.5%) for 5 times and incubated with tetramethylbenzidine (TMB) colorimetric reagent for 5 - 15 min, then the colorimetric reaction was stopped with stop solution ( HCI 0.1 %). The result of the reaction was read on a TECAN Sunrise plate reader, using a 450nm filter and the data was exported to be calculated and plotted after triplicating the reaction.

[0092] The values of immunoglobulins (IgA, IgM and IgG) present in milk after the different freeze-drying processes are described in Figure 1, as well as the degree of retention (%) of immunoglobulins when compared to the initial amounts present in the milk sample frozen human (control).

[0093] It was not possible to analyze the fluid milk due to the time required for transportation and analysis, as it is an extremely sensitive material under ambient conditions and with limited time in the refrigerator. For this reason, it was decided to use frozen milk as a control sample.

[0094] Freezing milk is the method commonly used in Milk Banks for storing and transporting samples. Human milk that is administered in ICUs goes through the freezing process at some stage of its cycle, from donation to the moment of administration. The use of frozen milk as a control sample is the closest condition to reality. [0095] During the drying process, 3 (three) energies are involved in the process: radiation, conduction and convection. Radiation consists of the transfer of heat through radiation from the walls and shelves of the equipment; conduction, in the transfer of heat through direct contact between the shelf and the sachet and between sachets, and, finally, convection is the transfer of heat through the convection of particles present in the chamber. Among the energies involved in this stage, convection is the main form of heat transfer. The particles in constant movement and collision between said particles and the sachets generate heat exchange. Therefore, a reasonable vacuum pressure is desirable for adequate convection to occur.

[0096] During the experiments, it was found that the use of a greater vacuum tends to preserve immunoglobulins more during the freeze-drying process when compared to higher values.

[0097] Surprisingly, it was observed that, in some samples, freeze-dried human milk has a greater amount of immunoglobulins when compared to the control sample, frozen human milk. This fact may be due to the storage of the control sample in a freezer, a method commonly used to preserve milk. Standard freezing, at -20°C, is ideal for short-term storage of samples that are not stable at higher temperatures. In this freezing process, the cooling speed is slow when compared to equipment with lower temperatures, resulting in larger ice crystals, which form over the time the material is stored and which can damage the milk components. In these cases, the external water solidifies before the interstitial medium, as it is less concentrated, corroborating possible damage to some components of the milk due to the difference in vapor pressure. It is worth remembering that conventional freezing equipment may also have an automatic defrosting function to prevent the accumulation of ice in the equipment. Temperature variations can also impact the sample components.

[0098] When human milk is freeze-dried, the amount of water in the sample is reduced, minimizing possible degradation reactions, as well as impacts related to the size and shape of ice crystals resulting from prolonged storage of the frozen sample.

[0099] Since IgG is found in low quantities in human milk, it was found that in some tests this immunoglobulin was below the method's quantification limit, or presented a high standard deviation between analyses, making comparative analysis difficult. of IgG in some cases. However, the gain obtained in preserving IgA and IgM immunoglobulins when using a higher vacuum pressure is clear.

[0100] It is worth mentioning that several factors influence the amount of immunoglobulin produced in the milk of lactating women, such as diet, period of breastfeeding and external factors. Therefore, each experiment was carried out with the same batch of milk.

[0101] Surprisingly, it was found that the temperatures used in the experiment do not have a significant impact on the degradation of immunoglobulins and that the vacuum used in the process is the most important parameter for the preservation of immunoglobulins in freeze-dried milk.

EXAMPLE 2 - Human milk freezing process using different methods.

[0102] The thawed pasteurized human milk was packaged according to the procedure described in Example 1.

[0103] Milk freezing was carried out using different methods, all listed in Table 2, such as freezer (P1 F), ultrafreezer (P1 UF) and liquid nitrogen (P1 N), according to the following conditions.

Table 2. Milk freezing methods

[0104] For freezing in freezers and ultrafreezers, the milk cooling rate was monitored in the different equipment according to data gathered in Table 3:

Table 3. Monitoring the cooling rate of milk during freezing in freezers and ultrafreezers

[0105] The milk frozen by the methods described was subjected to the same freeze-drying cycle, under the parameters in Table 4 below:

Table 4. Freeze drying parameters of frozen milk

[0106] The human milk powder obtained after freeze-drying was reconstituted and analyzed according to Example 1.

[0107] The values of IgA, IgM and IgG immunoglobulins in mg/mL are described in Figure 2. The immunoglobulins (lg) were quantified individually. Additionally, they were added together to obtain the amount of total lg immunoglobulins (IgA, IgM and IgG) in the samples, shown in Figure 3.

[0108] According to the total immunoglobulins evaluated, the freezing method using ultrafreezer showed promise, just like the method using liquid nitrogen, since the standard deviation obtained during sample analysis must be considered. However, evaluating individual quantifications, the results indicate that the method using ultrafreezer is the one that best preserves immunoglobulins, especially IgA, among the methods tested.

[0109] In deep freezing, high cooling rates are obtained, resulting in smaller ice crystals that do not damage the structure of the components. Furthermore, the external water and the interstitial medium solidify at the same time, reducing the vapor pressure difference between them.

[01 10] To corroborate the results found by the method above, the ion exchange purification technique was used. Milk samples of 0.5ml_ were analyzed by ion exchange chromatography on an Anionic Q-Sepharose™ column, pre-equilibrated with buffer A (20mM monobasic sodium phosphate at pH 6.3) and eluted in buffer B (20mM monobasic sodium phosphate with pH 7.0, 1M sodium chloride). The corresponding fractions were pooled by eluting buffer A and eluting buffer B. The total protein in the eluate was quantified using the BCA assay colorimetric method and evaluated on SDS-PAGE gel, as shown in Figure 4.

[01 11 ] Also using this technique, among the methods tested, the results indicate that the method using ultrafreezer is the one that best preserves the total proteins of freeze-dried human milk.

[01 12] Another relevant point for the freezing process is the issue of freezing and thawing the sample. During the experiments it was found that a single freezing is more effective when compared to milk samples that were thawed and frozen again more than once.

EXAMPLE 3 - Sample preparation and packaging process.

[01 13] After defining the freezing method and freeze-drying parameters, there was an opportunity to improve the preparation and packaging of samples.

[01 14] The fluid milk was packaged in aluminum sachets composed of polypropylene, polyethylene and other additives free of Bisphenol A, also known as BPA, with a parabola bottom, to avoid residues of freeze-dried milk in the corners of the sachet.

[01 15] 10mL of milk was filled in each sachet. These sachets were subjected to the freezing process in the Sanyo ultrafreezer model MDF-U33V with a temperature set point of -70 °C for 1 hour and freeze-dried on the shelves of the SP Scientific LyoStar3 freeze dryer with drying temperatures of 20 °C and 35 °C and vacuum pressure of 150pbar, according to Process 1 of Example 1 and freezing method 2 (P1 UF) of Example 2.

[01 16] Figure 5 shows the comparison of the concentration of IgA, IgM and IgG and retention percentage for frozen milk and freeze-dried milk samples. According to the results presented in this figure, it can be said that the process described in the invention is capable of preserving the immunoglobulins present in milk with retention rates greater than 84% and preferably greater than 90%.

[01 17] In this experiment it is evident that the freeze-drying process results in a product closer to fluid human milk when compared to frozen milk.

[01 18] The better performance in lyophilized samples, with regard to the preservation of IgA, IgM and IgG immunoglobulins, compared to frozen samples, may be due to the fact that during the freezing time of biological materials, it is known that ice crystals have specific morphologies that continue to grow over time and tend to damage sensitive materials, as explained in Example 1.

[01 19] In the proposed process, using rapid freezing in an ultrafreezer with subsequent removal of water, this event is mitigated.

EXAMPLE 4 - Assessment of the physicochemical and microbiological characteristics of freeze-dried human milk.

[0120] In addition to evaluating the immunological content of freeze-dried human milk, other quality parameters of powdered human milk were also evaluated, such as pH, reconstitution time, humidity, microbial count and presence of Bile Tolerant Gram-Negative Bacteria (Enterobacteriacea).

[0121] For the reconstitution time and pH analyses, the same samples were used.

[0122] The water used for analysis is ultrapurified and was previously heated to 40 °C to reconstitute freeze-dried human milk. The contents of the sachet were transferred to a beaker and 20mL of previously heated ultrapurified water were added, according to the initial filling volume. Time was counted using a stopwatch, which was started immediately after adding the entire volume of water. The sample was shaken and the powder dispersion was observed. The timer was paused only when all the powder was dispersed in the water and the time obtained was recorded. The values found are shown in Table 5.

Table 5. Results of reconstitution time, pH and humidity analyzes for freeze-dried colostrum and mature milk samples

[0123] With the milk already prepared for the reconstitution test, the sample went to pH analysis. The pH meter used was previously calibrated with at least two buffer solutions. The pH sensor was rinsed with water and then with a few portions of the material to be tested. The pH sensor was immersed in the material to be tested and the pH and temperature values were recorded. For all pH measurements, sufficient time was allowed for temperature stabilization and pH measurement. The results obtained from both the control sample and the milk sample after freeze-drying are shown in Table 5.

[0124] For humidity analysis, an infrared scale (with a halogen lamp source) was used at a temperature of 95 °C for 10 minutes. Approximately 1 g of sample was used and it was distributed evenly to cover the entire length of the aluminum tray of the scale to begin the measurement. The results are found in Table 5.

[0125] For microbiological analyzes, sachets were used that were filled with 30ml of frozen pasteurized human milk and subjected to the process of the invention. For microbial counting, the contents of the sachet (approximately 3g) were transferred to a bottle containing 30ml of sterile water and heated and homogenized until the sample was completely solubilized at a maximum of 40 °C. 10ml of the reconstituted milk was transferred to a bottle containing 90ml of the TA diluent, melted and maintained at 45-50T (Tryptone Azolectin Tween Broth) and homogenized (dilution 1:10). The microbial count (bacteria) was carried out using the plate counting method, transferring 1 ml of the 1:10 preparation to 2 Petri dishes. 15-20ml of Tryptic Soy Agar (TSA) medium was added, melted and maintained at 45-50°C, in 2 Petri dishes. The plates were homogenized carefully using “8” movements and the media was allowed to solidify. To count total aerobic microorganisms, the TSA plates (inverted) were incubated in an oven at 30-35 °C for 3 to 5 days. The plates were read, the arithmetic mean was calculated and multiplied by the dilution factor obtained. The value obtained is the number of CFU per ml_ of the product.

[0126] To search for pathogens, the procedure above was followed until the preparation of the diluted solution. It was homogenized and incubated at 22.5 ± 2.5 °C for two hours and no more than five hours (time necessary to reactivate the bacteria, but not enough to stimulate the microorganism to multiply). 1 ml of the diluted solution (1:10) was transferred to the Mossel Enterobacteriaceae Enrichment Broth. The preparation was incubated at 32.5 ± 2.5 °C for 24 to 48 hours. After this period, the subculture was prepared on plates containing neutral glucose red violet agar and incubated at 32.5 ± 2.5 °C for 18 to 24 hours. The sample meets the test if there is no colony growth. The microbiological results are shown in Table 6.

Table 6. Results of microbial count and presence of bile tolerant gram-negative bacteria for the mature milk sample before and after freeze-drying

[0127] The results obtained demonstrate that it is possible to obtain freeze-dried human milk with a quality similar to milk before freeze-drying. Just as the process proposed in the invention does not generate microbiological contamination.

[0128] To prepare freeze-dried human milk, an easy-to-use kit was developed for sending the material over long distances and places with a shortage of filtered water. This kit consists of an aluminum sachet with a parabola bottom, which prevents losses in the corners of the packaging, a 50ml bottle and lid with drinking water, a set of 10mL syringes and adapter, a packaging cartridge and a booklet containing how to prepare the milk, as can be seen in Figure 7.

[0129] It should be understood that the embodiments described above are merely illustrative and that any modifications to them may occur to a person skilled in the art. Accordingly, the present invention should not be considered limited to the embodiments described in the present report.

Claims

1. PROCESS FOR OBTAINING HUMAN MILK POWDER FOR THE PRESERVATION OF IMMUNOGLOBULINS, characterized by the fact that it comprises the steps of:

(a) fractionation of pasteurized human milk;

(b) freezing;

(c) drying; It is

2. PROCESS, according to claim 1, characterized by the fact that step (a) of fractionation comprises aliquoting 5 to 30 mL of human milk previously pasteurized at 62.5 °C for 30 minutes and filling into sachets.

3. PROCESS, according to claim 2, characterized by the fact that the sachets are aluminum and free from Bisphenol A.

4. PROCESS, according to claims 2 and/or 3, characterized by the fact that said sachets have a parabola bottom.

5. PROCESS, according to claim 1, characterized by the fact that step (b) of freezing human milk occurs in a temperature range of -80 °C to -30 °C, in an interval of 30 minutes to 5 hours.

6. PROCESS, according to claim 5, characterized by the fact that freezing step (b) comprises:

(ii) protect the top of the opened sachets with a plastic membrane.

7. PROCESS, according to claim 1, characterized by the fact that step (c) of drying occurs in a freeze dryer, in which the open sachets containing frozen milk are positioned on the shelves of said freeze dryer in a horizontal or vertical position, in a row or superimposed; primary drying occurs at 20°C and 150pbar; and secondary drying occurs at 35°C and 150pbar; and, wherein the residual moisture of the freeze-dried human milk powder is less than or equal to 5.0%.

8. PROCESS, according to claim 1, characterized by the fact that in step (d) the welding time of said sealing of the aluminum sachets is 2 to 8 seconds, with a welding power of 1 - 5 and in which the Said welding is preferably vacuum.

9. HUMAN MILK POWDER characterized by the fact that it is obtained by the process as defined in any of claims 1 to 8.

10. HUMAN MILK POWDER, according to claim 9, characterized by the fact that it comprises a degree of retention of IgA, IgG and IgM immunoglobulins greater than or equal to 84% in relation to frozen human milk.

11. KIT CONTAINING HUMAN MILK POWDER, in which the milk powder is obtained by the process as defined in claims 1 to 8, characterized by the fact that it comprises:

(a) a copy of the aluminum sachet with a parabola bottom containing freeze-dried human milk powder;

(b) bottle with a lid containing drinking water;

(c) syringe adapter;

(d) set of disposable syringes; It is

(e) cartridge for packaging materials.

12. USE OF THE KIT, as defined in claim 11, characterized by the fact that it comprises the steps of:

(a) open the kite and arrange the components for use; (b) preheat the flask containing drinking water in a water bath at 40 °C;

(c) transfer the contents of the sachet to the bottle of preheated water;

(e) check whether all content has been resolved;

(f) open the vial and position the syringe adapter

13. USE OF HUMAN MILK POWDER, as defined in claims 9 and 10 characterized by the fact that it is for the production of a food formula for babies and a reconstitution kit that protects against infections, diarrhea and allergies and promotes healthy growth, gain of weight and possible reduction in hospitalization time for premature babies.

14. USE OF HUMAN MILK POWDER, as defined in claims 9 and 10, characterized by the fact that it is for the manufacture of reconstituted human milk and a kit containing human milk powder, as defined in claims 1, 1 and 12.