The subject of the invention is a method of preservation for human milk, which enables quantitative reduction and elimination of pathogens in breast milk without damage to proteins and bioactive factors contained in that milk. The invention also relates to human breast milk preserved with this method.

BACKGROUND ART

Amongst currently used methods for human milk preservation the most common is heat treatment (pasteurization, sterilization). This preservation method reduces the microorganism levels, however it may result in degradation of valuable ingredients, such as inter alia antibodies, enzymes and vitamins.

The aim in the field is thus utilizing such a processing technology that in addition to microbiological safety would allow retaining natural or creating new, advantageous quality characteristics of the product, such as, e.g., bioactivity or stability, while minimizing undesirable changes caused by processing.

One of the techniques used is applying high pressure, so called pressurization, pascalization, high pressure processing or pressurizing. The high hydrostatic pressure technique (HPP), which is a modern technology for preservation and maintaining sensory characteristics of food, may be an alternative to heat treatment of many food products, as well as human breast milk. The use of high hydrostatic pressure in dairy industry has been known since the beginning of the century and was mainly focused on effective milk agitation process, i.e., homogenization, using a vacuum piston. More modern applications of HPP in dairy industry relate to the possibility of destroying harmful microorganisms. Subsequent years of the last century brought about solutions that allow obtaining food of a given microbiological purity and having nutritional value as well as taste qualities retained.

Currently, a dairy product has been marketed in Australia, which is subjected to high pressures, without additional heat treatment.

The use of this technology in relation to breast milk is associated with the development of milk banks and the necessity to ensure microbiological purity and therapeutic value of breast milk collected therein. First reports on the possibility of obtaining microbiologically pure breast milk upon pressurizing are from the beginning of the 21 st century (J Food Prot. 2008 Jan;71 (1 ):109- 18. Inactivation of bacterial pathogens in human milk by high-pressure processing. Viazis S, Farkas BE, Jaykus LA, High pressure processing of human milk for improved nutrient retention and microbial safety. Master Thesis North Carolina State University, 2006). In subsequent papers it was shown that the use of high pressure allows also to maintain ingredients of human breast milk that are valuable from the point of view of therapy in youngest patients (Molto- Puigmarti C, Permanyer M, Castellote Al, Lopez-Sabate MC. Effects of pasteurization and high-pressure processing on vitamin C, tocopherols and fatty acids in mature human milk. Food Chemistry. 201 1 :124 (3): 697-702, Viazis S, Farkas BE, Allen JC. Effects of High- Pressure Processing on Immunoglobulin A and Lysozyme Activity in Human Milk. J Hum Lact. 2007; 23( 3 ): 253-261 , Permanyer, M., Castellote, C., Ramirez-Santana, C., Audi, C., Perez- Cano, F. J., Castell, M., Lopez-Sabater, M. C. i Franch, A. (2010). Maintenance of breast milk immunoglobulin A after high-pressure processing. J Dairy Sci. 93(3):877-883).

Obtaining microbiologically pure breast milk with retained biological activity enables implementing patient feeding with species-specific milk in clinical practice, even when the mother does not have enough milk or when her food milk does not meet the nutritional needs of the baby. Such procedure involves supplementing mother's milk with milk from a milk bank, or fortifying baby's own mother milk with special formulas of known energetic value and nutrient content (so-called fortifiers). Currently operating milk banks (over 230 sites in Europe) use heat preservation - holder pasteurization (low temperature long time - LTLT) involving incubation of milk samples in 62.5 ^q C for 30 minutes. Temperature is measured inside a test sample, and is maintained by a thermostat, with heat-flow providing medium being water or air. The duration of pasteurization process is counted from the moment when the predetermined temperature in the final sample is reached and lasts for about 40 minutes. After 30 minutes of incubation rapid cooling occurs to 4°C (Wills M., Han V., Harris D., Baum J. (1982). Short time low-temperature pasteurisation of human milk. Early human development 7:71 -80). In doing so, microbiological purity of the product is ensured, but also multiple losses in its nutritional and therapeutic value are observed. Slightly better results in this regard are obtained when incubation time is reduced to a dozen or so seconds (15 sec) and the temperature is increased to 75 ^q C (high temperature short time - HTST). However, holder pasteurization is still the most popular method for mother's milk sterilization in hospitals (PeilaCh,Moro GE, Bertino E. The Effect of Holder Pasteurization on Nutrients and Biologically-Active Components in Donor Human Milk: A Review. Nutrients 2016, 8, 477;2-19).

Fortifiers available on the European market are produced from cow milk. The exception is US market, where Prolacta Bioscience fortifiers, derived from human breast milk preserved with a method using high temperature (HTST) are available. Although this type of preservation leads to significant decreases in the quality of the starting material for fortifiers production, clinical trials show that a diet based solely on breast milk, added as a supplement or a fortifier to mother's milk, brings significant clinical benefits in the group of most premature infants (Quigley M, McGuire W. Formula milk versus donor breast milk for feeding preterm or low birth weight infants. Cochrane Database of Systematic Reviews. 2014; Issue 4. Art. No.: CD002971 , Kantorowska A, Wei JC, Cohen RS. et. al. Impact of donor milk availability on breast milk use and necrotizing enterocolitis rates. Pediatrics. 2016;137:1 -8., Cristofalo EA, Schanler RJ, Blanco CL, Sullivan S. et al. Randomized trial of exclusive human milk versus pre-term formula diets in extremely premature infants. J Pediatr. 2013; 163. 1592- 5).

There are processes known in the prior art for preservation of human milk using high pressures.

The publication of Demazeau, Gerard et al.“A New High Hydrostatic Pressure Process to Assure the Microbial Safety of Human Milk While Preserving the Biological Activity of Its Main Components” Frontiers in public health vol. 6 306, November 6, 2018 describes a process for preservation of human milk using high pressures. A pressure of 350 MPa was used with the starting temperature of 38‘C, and decompression rate of 1 MPa.s ^-1 in 4 cycles of 5 min, with intervals of 5 min at atmospheric pressure. Good results in removing harmful microorganisms (including spores) were shown with simultaneous relatively good preservation of biological activity of milk components. However, certain disadvantage was indicated which was the duration of the process (approx. 90 min) combined with high equipment costs.

US patent application US 2015/0289530 A1 discloses a method for treatment of human milk, including a process to inactivate pathogenic factors and improve storage capabilities of the milk. The application discloses a method which includes subjecting human milk to at least two pressure pulses in the range of 200 to 400 MPa, with decompression rate between 0.5 and 100 MPa/s, and the initial temperature between 25 °C and 50 °C.

Wesolowska Aleksandra, Sinkiewicz-Darol Elena, Barbarska Olga, Strom Kamila Rutkowska Malgorzata, Karzel Katarzyna, Rosiak Elzbieta, Oledzka Gabriela, Orczyk-Pawitowicz Magdalena, Rzoska Sylwester, Borszewska-Kornacka Maria Katarzyna, 2018 New Achievements in High-Pressure Processing to Preserve Human Milk Bioactivity. Frontiers in Pediatrics, disclose a method for treatment of human milk using high pressure, in the following variants: (1 ) 600 MPa for 10 minutes; (2) 100 MPa for 10 minutes, interval for 10 minutes, 600 MPa for 10 minutes; (3) 200 MPa for 10 minutes, interval for 10 minutes, 400 MPa for 10 minutes; (4) 200 MPa for 10 minutes, interval for 10 minutes, 600 MPa for 10 minutes; at 19-21 °C. It was shown that in two out of four variants used, 200 MPa+400 MPa and 600 MPa, respectively, satisfactory microbiological purity of such treated milk was achieved (no aerobic mesophilic bacteria and Staphylococcus aureus·, although, no studies were conducted on fortified milk, but only on native flora), however, only the two-cycles variant, 200 MPa and 400 MPa, respectively, ensured maintaining high level of biological activity, including reducing the decrease of IgG, lactoferrin, leptin, HGF and insulin, IL-6, and erythropoietin levels. A decrease in adiponectin content was observed in all variants, although it was the weakest in the 200 MPa and 400 MPa variants.

The present inventors developed a new method for treatment of human milk, which is faster than the ones known in the art and requires lower energy input, but surprisingly allows to obtain milk, which is microbiologically safe, while maintaining very good biological properties. Therefore, the present invention relates to a method of preservation of human milk, comprising a step of subjecting milk to elevated pressure, wherein the pressure is in the range of 400-550 MPa, temperature is in the range of I d'Ό to Od'Ό, and milk pressure treatment is a one-step process - only a single pulse of pressure is used, with decompression time in the range of 80 ms to 1 s.

Therefore, the object of the invention is a method of preservation of human milk, comprising a step of treating milk with elevated pressure, wherein the pressure is in the range between 400 and 550 MPa, temperature is in the range of I d'Ό to 35 °C, wherein the step of treating milk includes treating milk using only a single pulse of pressure, with decompression time in the range of 80 ms to 1 s.

Fast decompression (i.e., in the range of 80 ms to 1 s) used in the method of the invention provides for especially effective microorganism eliminating activity (enabling potential reduction in the pressures used and shortening the duration of the process), but surprisingly, it does not lead to significant decrease in biological activity.

The inventors confirmed that when using pressure in the range of 400-550 MPa, excellent microbiological purity of such treated milk is achieved, despite using only a single pressure treatment cycle. Surprisingly, the method of the invention allows for the same reduction in pathogenic microorganism levels, while retaining much more bioactivity than pressure methods known in the art. Without wishing to be bound by theory, it can be concluded that a short decompression time after finishing pressurization is very important for the method of the invention. It is also not necessary to simultaneously use high temperatures. At the same time, the method of the invention provides human milk having very advantageous biological properties, since a biologically active form of essential biological molecules, such as proteins, e.g., HGF, insulin, leptin and/or lipase, IL-6, erythropoietin is retained.

In a preferred embodiment, pressure is in the range of 430-550 MPa, more preferably 450-500 MPa, particularly preferably is 450 MPa.

In a preferred embodiment, temperature is in the range between 15 and 30 °C, more preferably between 20 and 25 °C, and most preferably is 21 °C.

In a preferred embodiment, decompression time is in the range of 87-93 ms, and more preferably decompression time is 90 ms.

In a preferred embodiment, duration of treating milk with elevated pressure is between 5 and 25 minutes, preferably between 7 and 20 minutes, more preferably between 10 and 15 minutes, and most preferably duration of treating milk with elevated pressure is 15 minutes. Preferably, the method of the invention further comprises a step of lyophilization.

Preferably, the method allows to obtain milk having a shelf-life of at least 3 months in temperature from -18°C to 25°C. The object of the invention is also human milk preserved with the method of the invention, wherein the milk has HGF level of at least 70% of the content in raw milk and/or insulin level of at least 90% of the content in raw milk and/or leptin level of at least 90% of the content in raw milk and/or lipase level of at least 70% of the content in raw milk, and simultenously the milk does not exhibit any or has a level of microorganisms from the species of E. coli and/or S. aureus and/or L. monocytogenes and/or C. sakazakii ot below 10 ¹ cfu/mL

In a particularly preferred embodiment, the milk of the invention is lyophilized.

DEFINITIONS

The term ‘milk’ refers to a secretion produced by mammary glands of women and female animals, of a specific color, which is food for the newborn offspring.

The term‘lyophilization is intended to mean freeze-drying of frozen substances. With respect to‘lyophilized food’ the term relates to food preserved with the method of lyophilization, i.e., drying after freezing using vacuum.

The term‘elevated pressure’ in the meaning of the present invention means a pressure higher than atmospheric, e.g., in the range of several hundred MPa, e.g., 100-600 MPa, preferably, in the method of the invention 400-550 MPa.

The term‘bacterial titer’ refers to the number of bacteria in a given volume and is measured in colony forming units (cfu/mL).

‘Microbiological purity’ in the meaning of the present invention denotes an absence of a detectable amount or suitably low levels of pathogens, e.g., microorganisms from E. coli and/or S. aureus and/or L. monocytogenes and/or C. sakazakii species in the preserved milk. For example, the acceptable number of bacteria is <10 ² cfu/mL, more preferably <10 ¹ cfu/mL, the number of coagulase-positive staphylococci is, e.g., <10 ¹ cfu/mL, and the number of bacteria from the E. coli group is, e.g., <10 ¹ cfu/mL.

‘Raw milk’ denotes milk not subjected to heat treatment in elevated temperature or pressure treatment, and hence milk not subjected to the process of pasteurization, pascalization etc. ‘Fortified milk’ denotes milk intentionally infected with microorganisms in order to perform analyses of microbiological purity in challenge conditions with a known bacterial titer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the content of HGF (A), insulin (B), leptin (C) and adiponectin (D) in human milk treated with pressure, with the holder method or untreated (raw). The values for raw milk were taken as 100%.

FIG. 2 shows lipase enzymatic activity represented as a % of activity in raw milk.

FIG. 3 refers to the content of adiponectin (A), leptin (B), insulin (C), HGF (D), lactoferrin (E), and IgG (F) in human milk treated with the pressure, with the holder method or untreated (raw). The values for raw milk were taken as 100%. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for obtaining preserved human milk, with defined parameters of microbiological purity (preferably, a total absence of a detectable amount or suitably low levels of pathogens, e.g., microorganisms from E. coli and/or S. aureus and/or L. monocytogenes and/or C. sakazakii species in preserved milk, e.g., below 10 ¹ cfu/mL), but which has biological activity of milk components retained.

Milk samples were subjected to high pressure in the range of 400-550 MPa, for example, of 450 MPa, e.g., for 15 minutes, e.g., at an initial temperature of 20 °C, in the following controlled process parameters:

• medium temperature inside the pressure chamber,

• pressure profile, including its constant value at a given process pressure,

• time profile for increase and decline of pressure, along with the possibility of its sudden disappearance in a period within 0.1 s.

As a result of the preservation process, a material with retained therapeutic value was obtained, which was assessed using detection, unchanged in relation to untreated milk, of concentrations of the following factors: insulin, leptin, IL-6, erythropoietin, HGF, lipase. Selected factors represent milk components which are essential from the point of view of therapeutic significance, supporting the development of intestinal tissue and have a beneficial effect on digestive process through hormonal regulation.

Use of pressure in the range of 400 MPa - 550 MPa, more preferably 430-550 MPa, more preferably 450-500 MPa, particularly preferably 450 MPa in a single-pulse system unexpectedly allowed to obtain inactivation of S. aureus bacteria intentionally introduced to milk by 5 logarithmic orders. In addition, what should be emphasized, the effect of lack of coagulase production by the tested S. aureus strains persisted for 24 hours from the pressurization process.

Pressurizing human milk with the method of the present invention is effected using a processor generating high pressure. Such a device is known in the art and may consist of high-pressure chamber, a chamber sealing system (most often plugs reinforced by the frame of the device), a system for pumping pressure transferring medium through capillaries, and a system for operating and controlling the process. A very important element of the process system is a temperature stabilization system, which enables pressurizing in stable, repeatable conditions. The material subjected to high pressure pasteurization process must be placed in an airtight, flexible packaging, that allows for the pressure to be transferred to the material, human milk in this case. The packaging can be a polymer or composite bottle, a gas-tight polymer bag closed with a plug or heat-sealed. Then, the material in the packaging is placed in a pressure chamber. After closing the chamber, pressurizing process begins according to predefined parameters. Fast pressure changes, which are the most effective in their effect on pathogen cell membranes, are crucial. The minimum holding time, which allows the parameters to be improved, is 10 minutes at the maximum set pressure.

After the pressurizing process, the packagings are washed on the outside, and they are then stored below 20 °C.

Preservation with the method of the present invention was carried out in a high pressure processor. In addition, a safeguard was developed to prevent foreign solids from entering the processor system, involving sterilization of the system through an additional pascalization cycle with the aim to purify the medium before the actual process.

The analyzes showed that pressurizing in pulses (200 MPa 10 minutes / 10 minutes interval / +400 MPa 10 minutes and 200 MPa 10 minutes/ 10 minutes interval/ +100 MPa for 10 minutes, and 400/200 MPa for 10 minutes) is not effective for eliminating bacterial microflora in fortified milk, i.e., one infected with a known number of microorganisms. In turn, subjecting human breast milk to pressures of 600 MPa for 10 minutes does not guarantee any detection of components with characteristics of hormones and bioactive factors in sufficient quantities. However, it was unexpectedly shown that the milk preserved using the high pressure method in a variant comprising pressure range of 400-550 MPa, for a relatively short period, e.g., for 15 minutes, and particularly preferably at a pressure of 450 MPa, e.g., for 15 minutes, maintains a high concentration of bioactive ingredients, as well as ingredients with characteristics of hormones while ensuring elimination of pathogenic microorganisms. Surprisingly, it is sufficient to use a single pressure treatment cycle. It is particularly preferred to use a single cycle at a pressure of about 450 MPa, however the effect of eliminating microorganisms present in milk, maintaining bacteriostatic properties of milk and retaining biological activity of proteins using only a single pressure treatment cycle is achieved at a pressure within the range of 400-550 MPa.

EXAMPLES

Example 1

Pressurizing human milk

Pressurizing human milk was carried out using a processor generating high pressure. U4000/65 device (Unipress Equipment, Poland), with an active volume of up to V=2L, includes a high-pressure chamber (0,95 L), a chamber sealing system, a system for pumping pressure transferring medium through capillaries, and a system for operating and controlling the process. The pressure transferring medium is a mixture of water and polypropylene glycol in a 1 :1 ratio. A very important element of the process system is a temperature stabilization system, which enables pressurizing in stable, repeatable conditions. A safeguard was used, to prevent foreign solids from entering the processor system involves sterilization of the system through an additional pascalization cycle, with the aim to purify the medium before the actual process. The material subjected to high pressure pasteurization process was placed in an airtight, flexible packaging, with a volume of several ml. ³ , allowing for the pressure to be transferred to the material to be pasteurized, human milk in this case. The packaging can be a polymer or composite bottle, a gas-tight polymer bag closed with a plug or heat-sealed. Then, the material in the packaging was placed in a pressure chamber. After closing the chamber, the pressurizing process begun according to predefined parameters. Fast pressure changes, which are the most effective in their effect on pathogen cell membranes,, are crucial. The minimum holding time, which allows the parameters to be improved, is 10 minutes at the maximum set pressure.

The starting temperature was in the range of 19-21 °C.

After the pressurizing process, the packagings were washed on the outside, and then they were stored below 20 °C.

Different variants of pressure values, process times and the number of pulses (cycles) were used for the tests. The treated milk was subjected to microbiological tests (including fortified milk tests) (Example 2, points A, C, D) and bacteriostatic activity tests (Example 2, point B) as well as tests for the content of biologically active proteins (Example 3).

Lyophilization of milk

The process of lyophilization of human milk involving freezing water out by means of ice sublimation, was carried out by freezing fresh liquid raw material to -20 °C, followed by lyophilization at 30-40 °C in vacuum. The process of lyophilization was carried out for 24 hrs to 72 hrs under vacuum. Vacuum was achieved using pumps and freon. As a result of the process, a dry product was obtained, in the form of a powder.

Example 2

Microbiological tests of human milk subjected to high pressures and lyophilization

A) Milk treated with pressure (sinqle pulse of pressure of 450 MPa, 15 minutes) and/or lyophilized. Microbioloaical purity tests.

Human milk samples was collected from 5 donors, then stored in sterile polystyrene bottles at -20 °C. Thawed milk samples were used for the experiment. Milk was pooled with the same volume from each donor (1600 ml. x 5) to a final volume of 8000 ml. of raw milk. 7700 ml. of milk was used for microbiological tests. The remaining 300 ml. was a reserve.

From the total pool 7700 mL of combined raw milk, 100 ml. was left for microbiological analysis, to test for the presence of saprophytic bacteria and potential food-borne pathogens.

Microbiological tests were conducted in accordance with European standards for testing food products, for: total number of aerobic, mesophilic bacteria (Plate Agar Count, Merck, EN:ISO 4833÷2004), gram-negative intestinal E. coli bacteria from the family of Enterobactehaceae (Crystal-Violet Neutral Red Bile Glucose Agar, EN:ISO 21528÷2017), coagulase-positive Staphylococcus aureus (Baird Parker Agar, EN:IS06888- 35 3÷2004), gram-positive Listeria monocytogenes bacilli (Fraser Broth, EN:ISO 1 1290÷2:2017-2), gram-negative Cronobacter sakazakii bacilli (ESIA Agar, EN:ISO TS 22964÷2017), spore-forming bacteria Bacillus cereus (MYP Agar, EN:ISO7932÷2004).

Microbiological analyzes of raw milk were performed within 30 min from pooling. Milk was stored at 4°C.

The remaining volume of pooled (7600 ml.) raw milk was subjected to a standard long-term pasteurization process at 63 °C for 30 min, in sterile polystyrene bottles.

100 ml. of pooled pasteurized milk was subjected to microbiological analyzes, in regard to bacteriological purity of milk, and thus control of the pasteurization process. To that end, a test suite was performed to detect total number of bacteria and potential pathogens, according to the scheme: total number of aerobic, mesophilic bacteria (Plate Agar Count, Merck, EN:ISO 4833÷2004), E. coli (Crystal-Violet Neutral Red Bile Glucose Agar, EN:ISO 21528÷2017.), Staphylococcus aureus (Baird Parker Agar, EN:ISO 6888-3÷2004.), Listeria monocytogenes (Fraser Broth, EN:ISO 1 1290÷2:2017-2), Cronobacter sakazakii (ESIA Agar, EN:ISO TS 22964÷2017), Bacillus cereus (MYP Agar, EN:ISO 7932÷2004.).

Microbiological tests were performed within 30 min after pasteurization process was completed.

Milk was stored at 4 ^q C.

Preparation of bacterial strains

Bacterial strains were stored at -20 °C in 20% glycerol and in cryobanks (Argenta). Standard strains were grown in Brain Heart Infusion BHI liquid medium (Oxoid) at 37°C for 24 hrs. Then control cultures for strain growth were inoculated, from liquid cultures to selective solid media, as recommended in accordance with European standards for microbiological testing of food EN:ISO: E. coli (Crystal-Violet Neutral Red Bile Glucose Agar, E. coli Chromogenic Medium, 37^, 24 hrs), S. aureus (Baird Parker Agar, 37°C, 24 hrs, BHI Broth 37 ^q C, 24 hrs, Coagulase), L. monocytogenes (Half Fraser Broth 30 °C, 24 hrs, Faser Broth 37 ^q C, 48 hrs, Chromogenic Listeria 37°C, 24 hrs, Palcam 37°C, 24 hrs, TSEYA Agar 37 ^q C, 48 hrs, Rhamnose/Xylose test, Blood Agar 37°C, 24 hrs), Cronobacter sakazakii (CBS Broth 44 °C, 24 hrs, ESIA Agar 44 °C, 24 hrs, TSA Agar 37 ^q C, 24 hrs), Bacillus cereus (MYP Agar, Blood Agar, 37°C, 24 hrs).

Contamination of pooled pasteurized milk

The remaining 7500 mL of pooled pasteurized milk was stored at 4°C. The total volume was divided into 5 equal fractions 1500 mL each. Each portion of milk was contaminated with bacteria carried by food, including human milk. Inoculation of pooled pasteurized milk samples was carried out within 30 min after pasteurization process was completed. Pathogens that may cause gastrointestinal, respiratory and skin infections, as well as serious systemic infections, including meningitis and septicemia, in neonates and infants were selected for the experiment. Reference strains of four non-spore forming bacteria species were used: Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 33862), Listeria monocytogenes (ATCC 7644), Cronobacter sakazakii (ATCC 51329) and one species of spore-forming bacteria Bacillus cereus (ATCC 14579). Strains checked for characteristic growth were incubated in 500 ml. BHI liquid medium at 37°C for 24 hrs. Bacterial growth in culture broth was determined by measuring optical density of the culture by spectrophotometric method at 600 nm. Individual bacterial cultures were inoculated into separate pasteurized milk samples up to the final amount of 10 ⁶ cfu of bacteria in 1 ml. of milk. Infected milk was incubated at 37°C for 4 hrs, until stationary phase was reached. After incubation, from each inoculated milk sample, microbiological control cultures were made on propagating and selective media, in accordance with the above-mentioned standards.

Microbiological purity tests of milk after pascalization and lyophilization

After the incubation was complete, specific samples of inoculated milk were subjected to three processes: pascalization (HPP), lyophilization (LIO), and pascalization with subsequent lyophilization (HPP+LIO).

Pascalization process (pressure treatment) was carried out by the method of the present invention using a pressure of 450 MPa for 15 minutes, at an initial temperature of about 20 °C, a time profile with an increase and decline in pressure, along with the possibility of rapid decompression in the time of order of 0.1 s. Pascalization was performed in a chamber of U4000/65 device (Unipress Equipment, Poland).

The process of lyophilization of human milk, involving freezing water out by means of ice sublimation, was carried out by freezing fresh liquid raw material to -20 °C, followed by lyophilization at 30-40 °C in vacuum. The process of lyophilization was carried out for 24 hrs to 72 hrs under vacuum. Vacuum was achieved using pumps and freon. As a result of the process, a dry product was obtained in the form of a powder. The process was carried out in TG-30 vacuum apparatus (Germany).

For each process and each bacterial strain, experiments were performed in five replicates ([5 x 100 ml_] x 15]). For each of the variants, microbiological analyzes were performed after the physical process was applied. In order to determine the number of individual microorganisms in the samples, inoculations on liquid and solid media, propagating and selective, were done. Inoculations of milk samples were conducted in accordance with European standards for testing food products, for: total number of aerobic, mesophilic bacteria (Plate Agar Count, Merck, EN:ISO 4833÷2004), gram-negative intestinal bacteria E. coli from the family of Enterobactehaceae (Crystal-Violet Neutral Red Bile Glucose Agar, Coliform Chromagar, EN:ISO 21528÷2017.), coagulase-positive Staphylococcus aureus (Baird Parker Agar, BHI Broth, Coagulase, EN:ISO6888-3÷2004), gram-positive Listeria monocytogenes bacilli (Half Fraser Broth, Fraser Broth, Chromogenic Listeria, Palcam, TSEYA Agar, Rhamnose/Xylose test, Blood Agar, EN:ISO 1 1290÷2:2017-2), gram-negative Cronobacter sakazakii bacilli (CBS,ESIA Agar, TSA Agar, EN:ISO TS 22964÷2017), spore-forming bacteria Bacillus cereus (MYP Agar, EN:ISO7932÷2004). After performing individual physical processes, milk samples were initially diluted in peptone water and initial propagation of bacteria on liquid substrates was performed. Inoculations on selective and differential media were done by serial dilution method to determine the number of bacteria in 1 mL of the sample tested (cfu/mL). Microbiological inoculations of milk after HPP were performed within 10 hrs after the pascalization process was completed. Milk tests after LIO and HPP+LIO were performed according to the work schedule. Milk was stored at 4°C until testing.

Results

Table 1 below shows bacterial titers (log cfu/mL) in milk treated after inoculum was added and in the same milk after subjecting it to pasteurization with the method of the present invention using a pressure of 450 MPa for 15 minutes, at an initial temperature of 20°C, with a time profile with an increase and decline in pressure, along with the possibility of its sudden decline within the time of order of 0.1 s:

Table 1. Results of microbiological challenge tests for human milk subjected to pascalization (450 MPa, 15 minutes).

The use of preservation with a high pressure method with the pressure variant of 450 MPa for 15 minutes, combined with subsequent lyophilization process resulted in total destruction of vegetative forms of S. aureus, E coli, L. monocytogenes, and C sakazakii (Table 2). Table 2. Results of microbiological challenge tests for human milk subjected to pascalization (450 MPa, 15 minutes) and lyophilization.

It was demonstrated that the lyophilization process performed alone did not show the required effectiveness in terms of microbiological purity. Only for C. sakazakii, total destruction of vegetative forms was demonstrated. The results are shown in Table 3 below.

Table 3. Results of microbiological challenge tests for human milk subjected to lyophilization

B) Milk treated with pressure and milk treated with the holder method. Test for bacteriostatic properties

The following experimental samples were used to determine bactericidal properties:

· raw milk (not subjected to preservation processes),

• milk subjected to a traditional pasteurization method (holder method),

• milk subjected to the pasteurization method using high pressures in the conditions of 450 MPa, 15 minutes.

To perform assessments, the methodology described earlier by Silvestre et al. (Effect of pasteurization on the bactericidal capacity of human milk. Journal of Human Lactation. 2008;24(4):371 -6.) was used. The strain Escherichia coli NCTC 91 1 1 ((serotype 01 1 1 : K58 (B4): H-, Ogundele, 2002 - de la Coleccion Espanola de Cultivos Tipo (CECT, Burjassot, Valencia)) was used in the test.

Bacteria were grown overnight on nutrient agar, then suspended in peptone water to obtain a titer of 10 ⁸ cfu/mL. 0.2 mL of bacterial solution was added to 0.8 mL of individual samples, it was vortexed and incubated for 2 hours at 37°C. Control samples were 0.8 ml BHI (brain heart infusion) medium, to which 0.2 mL bacterial solution was added, and then incubated for 2 hours at 37 °C. Each sample and control samples were prepared in duplicates. After incubation samples were seeded at appropriate dilutions on VBRA (violet red bile agar), counted after incubation for 24 hours at 37 °C. Bactericidal properties were calculated as differences between the sample and the control sample and expressed in percentage. 100

NO - control sample

Nf - tested sample

The results are shown in Table 4 below as percentages relative to control sample (% reduction in growth of E. coli). Mann-Whitney U-test was used to assess statistical significance, considering p<0.05 as statistically significant. STATISTICA version 13.1 (Stat Soft. Inc.) was used for data analysis.

Results:

Pasteurization by the holder method was shown to reduce with statistical significance the bactericidal properties of human milk relative to raw milk (12.12% vs 46.60%). The reduction of bactericidal properties of human milk subjected to pasteurization by the high pressure method was not statistically significant, which means that the pascalized milk has retained bactericidal properties.

Table 4. Bactericidal properties of milk treated with the method of the invention (single pulse of pressure of 450 MPa, 15 minutes) and milk treated with the holder method.

C) Milk treated with pressure in several one- and two-pulse variants.

The material for study was frozen, unpasteurized breast milk obtained from the Regional Human Milk Bank (Regionalny Bank Mleka Kobiecego) in Warsaw in 2016. Before contamination, the milk was thawed at 40°C in water bath (Danlab). After thawing, milk obtained from 3 donors was pooled.

Selection of Staphylococcus aureus strains.

Three strains of Staphylococcus aureus bacteria were selected as indicator microorganisms: ATCC 25923 commonly used in studies of the inactivating effect of high pressures, 4.4. isolated from food from the Collection of the Department of Industrial Microbiology of Food (Katedra Mikrobiologii Przemystowej Zywnosci) of the University of Warmia and Mazury, HM01 strain isolated from unpreserved breast milk at the Food Hygiene Institute (Zaktad Higieny Zywnosci) of SGGW in Warsaw. The purpose of using a cocktail of various strains of S. aureus bacteria was to check the effect of high hydrostatic pressure on the microflora potentially found in milk derived from several women. Before being used for contamination of milk, each S. aureus strain was subcultured three times, obtaining each time an 18-24 hour culture at 37 °C. Bacterial cells were centrifuged twice at 12,000 x g for 4 min and then suspended in 0.1 % peptone water. The resulting microbial suspension had a final concentration of 10 ⁹ cfu/mL.

Milk contamination

Contamination of milk was carried out under sterile conditions by adding 3 ml. of each strain to a thawed 150 ml portion of milk to obtain a final concentration in the order of 10 ⁷ cfu/mL. Contaminated milk was poured into sterile bottles commonly used during the pasteurization process (Sterifeed) and into bottles of capacity 30 ml. made of high density pressure resistant polyethylene (Bionovo). In parallel to the preserved milk samples, test in a control sample K (unpreserved milk contaminated with S. aureus bacterial cocktail) was carried out. Three replications of a 14-day storage cycle of human milk contaminated with a cocktail of S. aureus bacteria inactivated by pasteurization and high pressure were performed in several pressure and time variants.

Research methods

Microbiological analyzes

Microbiological analyzes were carried out using the classical plate method according to EN ISO 6888-2:1999. The following media were used for the tests: Barid-Parker with supplement (Labo) and nutrient agar prepared in accordance with the manufacturer's instruction. The necessary decimal dilutions of the samples were made using 0.1 % peptone water. After microbiological seeding, the plates were incubated at 37 °C for 24-48 hours. Grown microorganisms were counted using a colony counter (PolEco). Black and steel-colored colonies surrounded by a transparent zone were counted according to the manufacturer's instruction (Labo)

A 14-day storage period for milk preserved with various methods was used. The day when the milk was contaminated and subjected to preservation by pasteurization and pressure was marked as day "0". Then microbiological analyzes were performed after 1 , 2, 4, 9 and 14 days of storage. After the test, all test and control samples were stored in refrigerated conditions (temperature 4 ^q C) while maintaining cool chain during transport of samples from the laboratory to the Regional Human Milk Bank and the Institute of High Pressure Physics of PAS. Results

Comparative studies were carried out in the following experimental variants:

a. single-pulse pressure of 400 MPa or 450 MPa for 10 min

b. pressure in two-pulse configuration, in sequence of 200 MPa and 400 MPa, respectively, and in sequence of 400 MPa and 200 MPa, respectively for 10 min, with 10 min interval

The results are shown in Table 5 below.

Table 5. Results of microbiological tests for fortified human milk subjected to pascalization in various pressure variants.

In the case of a single-pulse pressure of 400 MPa, 10 min after the milk preservation process, on average 2 log cfu/mL of the S. aureus strain cocktail used for contamination was measured. With respect to milk pressurized in pressure conditions of 450 MPa for 10 min, after the milk preservation process, as well as during storage period, S. aureus and Cronobacter spp. bacteria (native microflora) were not detected.

In another experimental variant, two-pulse pressure with a pressure of, sequentially, 200 MPa, 10 min, 10 min interval and 400 MPa, 10 min, total inactivation of native milk microflora was achieved, however it was demonstrated that only 58.6% S. aureus strains were inactivated. Similarly, the experimental variant comprising two-pulse pressure with a pressure of, sequentially, 400 MPa, 10 min, 10 min interval and 200 MPa 10 min, resulted in inactivation of 40.4% S. aureus strains used for prior contamination.

D) Milk treated with pressure in several one- and two-pulse variants. Studies on the effectiveness of elimination of endoaenous bacterial flora

Human milk samples from 80 donors from the Regional Human Milk Bank in Warsaw (~50 ml.) were stored in 4°C. To obtain a minimum volume of 125 ml. for the study, samples from 2-4 donors were pooled. Each pool was aliquoted and subjected to pressurizing, as described in Example 1 , in 4 variants listed below.

The starting temperature was 19-21 °C. The time to generate pressure was 15-25 seconds, decompression time was 1 -4 s. The following variants were tested:

(1 ) 600 MPa, 10 minutes;

(2) 200 MPa, 10 min; 10 minutes interval; 400 MPa, 10 minutes;

(3) 100 MPa, 10 min; 10 minutes interval; 600 MPa, 10 minutes;

(4) 200 MPa, 10 min; 10 minutes interval; 600 MPa, 10 minutes. As a control, aliquots of milk were subjected to pasteurization using the holder method in Sterifeed S90 ECO Pasteurise device (Medicare Colgate Ltd.), at 62.5 ^q C for 30 min.

Microbiological analyses

Microbiological analysis was carried out to confirm microbiological purity of the samples. The analysis was performed in triplicate, for the total number of aerobic mesophilic microorganisms (PN-EN ISO 7218: 2008 / A1 : 2013, PN-EN ISO 6887-5: 2010) and the number of Staphylococcus aureus (PN-EN ISO 6888-1 : 2001 / A1 : 2004).

Results

The results of microbiological purity tests for treated milk samples, as in Example 1 are shown in Table 6.

Table 6. Microbiological purity of human milk samples subjected to pascalization in various pressure variants.

Pressure-treated milk in each variant, as well as the control milk (subjected to pasteurisation) was microbiologically pure.

However, it should be noted that the variant (2), 200 MPa, 10 min / 10 min interval / +400 MPa, 10 min, was also tested in challenge tests, with intentional infecting of milk samples before the pressurizing process (see Table 5 above) and total elimination of pathogenic microorganisms was not obtained then. Unexpectedly, treating with pressure in just one cycle is visibly more effective.

Example 3

Determination of biological activity of milk subjected to high pressures and lyophilization

A) Milk treated with pressure and milk treated with the holder metod. Determination of biological activity.

In milk samples subjected to pressure treatment, according to the methodology described in the preceding examples, content and biological activity were determined for several essential components of human breast milk:

i) determination of HGF, insulin, leptin, adiponectin content, and

ii) lipase activity testing

in milk samples pressurized 450 MPa, for a period of 15 minutes and lyophilized. In order to determine biological activity, 6 samples of pooled milk from donors were used. Each sample was combined milk from 3-4 donors, which was then divided into smaller portions and subjected to various preservation processes. The experiment was repeated 6 times. To confirm the suitability of the experimental variant at a pressure of 450 MPa, 6 samples were analyzed in 3 different variants:

• raw milk (not subjected to preservation processes),

• milk subjected to traditional pasteurization method (holder method: in Sterifeed S90 ECO Pasteurise device (Medicare Colgate Ltd.), at 62,5°C for 30 min),

• milk subjected to pasteurization method using high pressures in pressure conditions of 450 MPa, for a period of 15 minutes (see also description in Example 1 ),

• milk subjected to pasteurization method using high pressures in pressure conditions of 450 MPa, for a period of 15 minutes (see also description in Example 1 ) followed by lyophilization (description in Example 1 ).

After carrying out the above processes, all samples and raw milk were centrifuged - 4400 rpm for 15 minutes at 4 ^q C (Centrifuge 5702R, Eppendorf), for further analysis, the supernatants were separated in portions into Eppendorf type tubes and frozen at -21 °C.

The ELISA method was used to assess the content of bioactive ingredients. Leptin, adiponectin, hepatocyte growth factor (HGF) and insulin concentrations were analyzed using commercial ELISA kits, i.e., Human Leptin; Human HMW Adiponectin/Acrp30 (R&D Systems, Inc); Human HGF (R&D Systems, Inc); Insulin ELISA (DRG Instruments GmbH, Germany). Lipase activity was tested with a colorimetric test lipase assay kit (MAK046 Sigma-Aldrich).

Results

Milk preserved with a high pressure method in a pressure variant of 450 MPa for 15 minutes, and then subjected to lyophilization process retains higher concentration of bioactive ingredients than milk pasteurized with the holder method, respectively HGF 79.2 % in pressurized milk and respectively 72% in milk after lyophilization vs. 3.2% in milk after holder pasteurization; insulin 109% in milk after pressurizing and respectively 93% in milk after lyophilization vs. 90% after holder pasteurization, leptin 101 .4% in pressurized milk, 1 16% in milk subjected to lyophilization vs. 34.2% after holder pasteurization. For adiponectin a significant decrease in hormone content was observed after pressurizing 14.8% compared to 76% after holder pasteurization, which did not change after lyophilization 14.2% compared to raw milk. The results are shown in FIG. 1 (A-D).

It was found that milk preserved using the high pressure method in the pressure variant of 450 MPa for 15 minutes, retains almost unchanged lipase enzymatic activity at the level of 78% activity in pressurized milk, 75% in pressurized milk and subjected to lyophilization respectively compared to 3.8 % in milk after holder pasteurization. The results are shown in FIG. 2. B) Milk treated with pressure in several one- and two-pulse variants. Determination of biological activity.

Human milk samples from 80 donors from the Regional Human Milk Bank in Warsaw (~50 ml.) were stored at 4°C. To obtain a minimum volume of 125 ml for the study, samples from 2-4 donors were pooled. Each pool was aliquoted and subjected to pressurizing, as described in Example 1 , in 4 variants listed below.

The starting temperature was 19-21 °C. The time to generate pressure was 15-25 seconds, decompression time was 1 -4 s. The following variants were tested:

(1 ) 600 MPa, 10 minutes;

(2) 200 MPa, 10 min; 10 minutes interval; 400 MPa, 10 minutes;

(3) 100 MPa, 10 min; 10 minutes interval; 600 MPa, 10 minutes;

(4) 200 MPa, 10 min; 10 minutes interval; 600 MPa, 10 minutes.

As control, aliquots of milk were subjected to pasteurization with holder method in Sterifeed S90 ECO Pasteurise (Medicare Colgate Ltd.) device, at 62.5 °C for 30 min.

After carrying out the above processes, all samples and raw milk were centrifuged - 4400 rpm for 15 minutes at 4°C (Centrifuge 5702R, Eppendorf), for further analysis the supernatants were separated in portions into Eppendorf type tubes and frozen at -21 °C.

The ELISA method was used to assess the content of bioactive ingredients. Leptin, adiponectin, hepatocyte growth factor (HGF) and insulin concentrations were analyzed using commercial ELISA kits, i.e., Human Leptin; Human HMW Adiponectin/Acrp30 (R&D Systems, Inc); Human HGF (R&D Systems, Inc); Insulin ELISA (DRG Instruments GmbH, Germany). For the determination of lactoferrin, anti-human lactoferrin monoclonal antibody was used (ABCAM, Cambridge, UK) as a coating agent for wells of a microtiter plate (Nalge Nunc International, Naperville, II, USA) to bind lactoferrin from the sample. 100 pi of 5000-, 10 000-, 25 000- and 50 000-fold diluted milk and standard preparation of lactoferrin from 0.8 to 25 ng/100 mI (Sigma, St. Louis, MO, USA) were used for the tests. The amount of bound lactoferrin was quantified using phosphatase-labeled rabbit anti-human lactoferrin antibodies (Jackson ImmunoResearch, USA).

The IgG concentration was determined according to the following procedure: the fragment F(ab’) ₂ of goat anti-human IgG antibody (Jackson ImmunoResearch, USA) was used to coat wells of a microtiter plate (Nalge Nunc International, Naperville, II, USA) to bind IgG from the sample. 100 mI of 100-, 25-, 500- and 1 000-fold diluted milk and standard IgG preparation from 0.2 to 12.5 ng/100 mI (Jackson ImmunoResearch, USA) were used for the tests. The amount of bound IgG was quantified using phosphatase-labeled rabbit antibodies specific for the human IgG Fey fragment (Jackson ImmunoResearch, USA).

The result for the lactoferrin and IgG tests were read using 4-nitrophenyl phosphate (SERVA, Heidelberg, Germany) as a substrate, and the absorbance was measured in Stat Fax 2100 Microplate Reader (Awareness Technology Inc., Palm City, FL, USA) at 405 nm (reference: 630 nm). All binding and washing steps in ELISA tests were performed in TRIS buffered saline (TBS, pH 7.5) containing 0.2% Tween 20. All samples were analyzed in four different dilutions, each in duplicates.

Results

The results are shown in FIG. 3 (adiponectin (A), leptin (B), insulin (C), lactoferrin (E) and IgG

(F)).

For HGF the level 1 1.28 % was obtained (holder method), 36.15% (600 MPa), 38.81% (100 MPa + 600 MPa), 97.15% (200 MPa + 400 MPa) and 43.02% (200 MPa + 600 MPa) (FIG. 3 (D)).

SUMMARY

The results above show that treating human milk using the method of the invention, i.e. treating with pressure in the range of 400-550 MPa in a single cycle with rapid decompression, is unexpectedly sufficient to eliminate microorganisms (providing even better results in challenge test than when two cycles in this pressure range are used), while ensuring retaining biological activity of the milk components.

The present inventors have surprisingly found that using only a single pressure treatment cycle, in the range of 400-550 MPa, with decompression time in the range of 80 ms to 1 s allows to obtain an optimal effect, both in terms of microbiological safety and retaining biological activity of human breast milk. It is therefore possible to preserve human breast milk in a much simpler and shorter process than known in the art.