Comments on the High Pressure Preservation of Human Milk

The current state of studies on the high pressure preservation of the human milk is briefly presented. It is indicated that reaching (i) the antimicrobial safety, (ii) antiviral safety, and (iii) high nutritional, metabolic and immunological quality, may be difficult for a “classical” single pressure pulse High Pressure Preservation (HPP) treatment. It is shown that the sudden decompression leads to additional physical processes, which can be important for supporting the HPP technology. Additional advantages were reached due to the two-pulse compression, with subsequent values: P = 200 MPa and 400 MPa. Tests included the microbiological insight for the two-weeks storage. It is also shown that the decay of the number of microorganisms under the high pressure follows the relation n(t) = n0exp(At)exp(Bt ). Finally, issues regarding containers for the high pressure preservation of human milk are discussed.


Introduction
The human milk is not only the first food for new born infants. It is the natural continuation of the intrauterine nutrition which delivers the energy and enables the development. Human milk is essential for avoiding many chronic diseases during the whole life, being the source of constituents important in metabolic processes. For instance, epidemiological data show the protective role of the human milk to prevent acute and chronic diseases and obesity. If the breastfeeding of newborn is not possible, the best solution seems to be banks of human milk (Mimouni et al., 2017). Nowadays, the microbiological safety in human milk banks is assured via the Holder "soft" pasteurization: the thermal treatment at T = www.scholink.org/ojs/index.php/fsns Food Science and Nutrition Studies Vol. 1, No. 2, 2017 72 Published by SCHOLINK INC. 62.5 o C for t = 30 min (Kim & Unger, 2010). However, any protocol associated with approaching or passing the pasteurization threshold, required for reaching the microbiological safety, is associated with the very notable reduction of key nutritional and bioactive features (Peila et al., 2016). The New Hope for banks of human milk can be the implementation of innovative methods based on non-thermal physical treatments (Peila et al., 2017). The most prominent candidate seems to be the High Pressure Preservation (HPP), which has already reached a great success in the pro-health foods market. The key features of the HPP technology can be concluded as follows (Boziaris, 2014;Huang et al., 2016;Barba et al., 2017): One can expect that these extraordinary advantages of the HPP technology have to occur also for the human milk. However, human milk is much more than simply a food. It is an extremely complex system, being also one of the most important factors shaping the human health for the whole life (Mimouni et al., 2017). Consequently, possible implementations of the HPP technology for human milk banks require careful optimization and consideration of all possible issues.
This report discusses problems of the HPP technology application for the human milk. It focuses on physical issues which have been not clearly addressed so far, including compressing protocols and the convenient packing.

Method
New experimental results presented in this report are based on the application of large volume high pressure processors, working up to P > 600 MPa and in the possible temperature range from -20 o C to 120 o C. The working volumes of pressure chambers: V = 1 L and V = 2 L They enable "programming" of the form of high pressure pulses (see the description below) and measurement of temperature via thermocouples located within the chamber. Both processors were designed and manufactures in the the high pressure treatments the human milk samples were place in special container able to 20%-30% decrease of volume without any permanent and non-reversible distortions. All details regarding containers and microbiological tests are given is subsequent paragraphs.

Emerging Optimal Parameters of HPP Processing of Human Milk
Generally, studies of the high pressure based preservation of human milk explore the pattern developed for the food industry: the high compression in the range between 300 and 600 MPa, for few/several minutes, at the near room temperature. The key target in this treatment is the high microbiological safety (Huang et al., 2016). This pattern is also applied in studies on the human milk pascalization carried out so far (Vazquez-Landaverde et al., 2006;Viazis, 2008;Viazis et al., 2008;Permanyer et al., 2010;Moltó-Puigmartí et al., 2011;Lou et al., 2012;Delgado et al., 2014;Sousa et al., 2014;Windyga et al., 2015;Sousa et al., 2016;Kiełbratowska & Kołodziejska, 2017;Li et al., 2017). Tables 1 and 2 below illustrate some recent results, focusing on trends and the scatter of experimental data.   Note. Prepared using results of ref.
Recently, HPP studies of human milk focused on the impact of the sequence of high pressure pulses (lasting 6-10 minutes), with respect to nutritional, immunological and metabolic factors have been carried out (Girard et al., 2016). Their results are concluded in Table 3. The emerging picture from above results can be concluded as follows:  The post-HPP treatment nutritional, metabolic and immunological features are close to the native human milk, whereas the thermal HPP treatment is notably destructive. It seems that the optimal values of pressure is located for P = 300-400 MPa, i.e., in the region where the deactivation of pathogenic bacteria decreases.


The high pressure pascalization offers much better microbiological safety regarding key pathogenic bacteria and the total bacterial load. This target can be reached for P = 400-500 MPa. Worth stressing is the notable scatter of results obtained in different laboratories.


Regarding pathogenic viruses the evidence is still limited. Notwithstanding, one can expect that at least in some cases the safety is reached for P >> 600 MPa. This range of pressures is highly destructive for nutritional, metabolic and immunological factors of human milk.

The Form of High Pressure Pulses
For the high pressure "cold pasteurization" of foods, products are placed within the pressure chamber in a container capable for the reversible elastic deformation caused by the isotropic shrinking during process. In our studies two HPP processors, designed and build in IHPP PAS, with volumes of pressure chambers V = 2L (2r = 100 mm) and V = 1L (diameter 2r = 65 mm) were used. For the first processor (type I) the piston method for high compression was used (Yordanov & Angelova, 2010). In this case the durations of compressing ( rise t  ) and decompressing ( decay t  ) is obligatory in the range of 1-2 minutes. The pattern of high pressure pulse is shown in Figure 1. For the second processor (type II) the high compression was created due to the external pumping system, supported by the pressure mutiplicator. For the possible profile see Figure 2.
The associated changes of temperature are also given (red, dashed curve and the right scale). This is related for type I HPP processor with the piston-manostatic principle, volume V = 2 L, diameter 2r = 100 mm. Fast increasing/decreasing of pressure is coupled to notable changes of temperature, as shown in Figures 1 and 2 (dashed curves in red). This is associated with the adiabatic nature of the process, most often recalled within the ideal gas equation frames: , where P, T and V stand for pressure, temperature and volume; n is the number of moles and R denotes the gas constant (Stanley, 1972;Rzoska et al., 2010). Following this, for adiabatic heating/cooling associated with compressing/decompressing one obtains is the ratio of specific heats for constant pressure and volume. Such process is isentropic (the entropy S = const) and it is associated with the notable change of the internal energy  The associated changes of temperature are also given (red, dashed curve and the right scale). The plot is for the type II HP processor with the external pumping of pressure, volume V = 1 L, diameter 2r = 65 mm. The temperature sensor was located on the axis of the chamber, 20 mm below its top.
It is worth stressing that the adiabatic change of pressure and temperature occurs "immediately" and homogeneously within the whole processed sample/material. Ideal adiabatic conditions appears for the perfect thermal isolation from the surrounding or for processes fast enough to reduce the heat exchange from/to surrounding. Above relations although "classical" in discussions on the adiabatic heating/cooling assume the ideal gas equation as the reference. Its validity is limited to gases since for the ideal gas the phase transition to the liquid state is absent (Stanley, 1971). For liquids, a dense and elastic medium, notable repulsive and attractive interactions are significant. Consequently, one can explore solely the isentropic nature of the adiabatic compression/decompression the nature of the given phenomenon, namely: where P  is for thermal expansion coefficient, P c denotes the isobaric  is for density (Stanley, 1971;Rzoska et al., 2010).   = 600 MPa pressure jump, assuming water as the host pressurized medium. Notable is the similarity of the above dependence to the 2 nd Ehrenfest equation (Stanley, 1971;Rzoska et al., 2010), used for describing the pressure evolution of the phase transition: the rapid change of pressure is mostly adiabatic but some heat exchange also can take place. This can lead to the supplementary change of entropy, related to the Clausius-Clapeyron equation (Stanley, 1972;Rzoska et al., 2010). Figure 1 shows the most classical form of HPP pressure vs. time pulse. First, the pressure increases (for s t rise 125   125 s), subsequently it is kept at the processing pressure for . stat t  (~ 5 min in Figure 1).

Containers for the HPP Treatment of Human Milk and Fluid Foods
For the HPP technology, products are placed within an elastic container, made from a plastic (polymer-based) material which does not interact with the product. The container has to ensure the isolation from the pressure-mediated liquid (water, often with lubricated additives as glycol) during the high pressure processing. It also must provide excellent pressure transmission to the product from the pressurized (liquid) medium and the return to the initial state after decompressing (Mertens, 1993;Balasubramaniam et al., 2016

Milk or Other Fluid Foods/Drinks
The solid (thick, blue) arrows indicates the (isotropic) way of compressing. The left bottle (A), made from an elastic plastic, enables HPP processing also in the case if the bottle is only partially filled with the fluid. The dashed (thick, blue) arrows show the additional "anisotropic" shrinkage due to the specific design. All these enable the "safe" compressing of a fluid "product" with a notable air layer inside the bottle. The right part of the figure (B) shows the possibility of making the bottle from an arbitrary material, including the silicate/oxide glass. The pressure is transmitted via increasing the volume of the internal "soft" immersed part (Rzoska & Drozd-Rzoska, 2017).
Generally, almost non-compressible glass bottles are considered as non-proper for the HPP technology.
On the other hand glass-bottles are multi-use, easy-cleaned and free from parasitic interactions with any food, including human milk. Figure 1b shows the possible solution of the "glass bottle problem".
The pressure is transmitted to the fluid by means of a properly shaped and "soft" element connected to the cap and immersed in the HPP processed liquid. This element can be also "reversed" and subsequently used as the bottle teat, if made from latex or similar a similar material.  (Balasubramaniam et al., 2016). A glimpse-test of Figure 4 suggests that such pattern obeys for T = 4 o C and T = 20 o C isotherms, but definitively fails for T = 50 o C.  In the basic case of the simple exponential decay one obtains:

Microbiological Tests of the New HPP Protocol
Such description is related to horizontal lines in Figure 5. However, it obeys only for T = 4 o C and T = 20 o C isotherms, for the limited period of time. Results presented in Figure 4 indicate the occurance of the more general pattern: Horizontal lines in Fig. 5  microorganisms is relatively mild, namely only 1.14 "log-unit" for t = 10 min compressing and 1.7 "log-unit" for t = 15 min.
The above resume of recent studies on the HPP technology for human milk focused on (i) microbiological safety, (ii) nutritional, metabolic and immunological agents, and (iii) anti-virus issues.
Consequently, the question arises how to overcome the problem of different optimal pressures for these key targets. A possible solution is a new compressing protocol. In ref. (Girard et al., 2016) the application of a sequence of up to 4 pressure pulses P = 400 MPa, duration 6-10 minutes each, for instance was tested. Results presented above indicate the necessity of the HPP+ application protocols for human milk, beyond the dominated "classical" HPP pattern, i.e., the single high pressure pulse. In this paragraph results of such HPP+ approach using 2 high pressure pulses are presented: (i) compression at P = 200 MPa for  t stat. = 10 min, (ii) for  t wait. = 10 min samples were kept at P = 0.1 MPa, and finally (iii) the compression at P = 400 MPa for  t stat. = 10 min: i.e., the same total period as in the Holder "standard" treatment. Such pattern was successfully tested (Skąpska et al., 2012;Skąpska et al., 2013) in studies on fruits and vegetable juices.   : the index D is for the given day and D = 0 stands for the reference onset day. For the reference see results of ref. (Windyga et al., 2015) where the single high pressure pulse for P = 400 MPa,  t stat. = 15 min at T = 20 o C yielded ca. 2-log decay of S. aureus.
Three replicates of a 14-day storage cycle of humane milk contaminated with Staphylococcus aureus inactivated by high pressure were made. The reference number of S. aureus determined in the control sample was changed significantly during the 9 days of refrigerated storage of milk samples. The number of bacteria recorded on that day was 4.43 log cfu/ml. Changing the number of bacteria by more than two orders of magnitude signified the natural aging of the population of the bacteria tested, as confirmed by the 14th day of storage, in which a 3.91 log cfu/ml was found. In 9 th day of the storage also in pressure samples (type II), the significant reduction in the tested microorganisms was recorded: down to a level of 1.79 log cfu/ml. However, the last day of the storage cycle was not labeled S. aureus in the pressurized milk samples. A similar phenomenon took place in the pressure samples in device I. The significant change in the number of bacteria compared to the reference sample was observed on day 4 of storage to level 1.75 log cfu/ml. Results presented below clearly show the increasing effectiveness of the proposed double-pulse high pressure pattern of compressing, reached already for the near-room temperature of processing T = 20 o C. The obtained result is qualitatively better than for the single pulse for P = 400 MPa processing at the near room temperature. The next notable result is much better reduction of HPP processing for the treatment with the "sudden" decay of pressure (the form of pulse presented in Figure 2). The application of the single 10 minutes, P = 600 MPa completely removes pathogens but it is strongly destructive basic functional features of the human milk. Taking into account results from Table 4 and ref. (Windyga et al., 2015) one can conclude the notable advantage of the 2-pulse scenario, with the properly selected values of compressing, supplemented by the sudden decay of the high pressure pulse.

Discussion
This report discusses selected issues associated with the high pressure "cold pasteurization" of human milk. The analysis of experimental data clearly shows that this non-thermal method may lead to results qualitatively better than obtained for the Holder thermal pasteurization, regarding the antimicrobial and antiviral safety as well as the preservation of bioactive nutritional, metabolic and immunological features. However, such completed preservation target seems to be not possible for the single pulse HPP technology. Such breakthrough target seems to be possible only for the expanded "HPP+" approach, such as discussed in this report 2-pulses protocol (P = 200 MPa and P = 400 MPa). For presented studies, it has been obtained that results of the treatment can remain valid for the 2-weeks storage. It is worth stressing that the deeper decrease of the number or S. aureus occurred for the sudden decompression of high pressure pulses. One can speculate that this is associated with the short-time, deep adiabatic cooling, the pressure-shock change and the pressure-gradient wave front propagated through the tested sample. The analysis of changes of the number of S. aureus under P = 400 MPa compression showed that the most often used near room temperature (T = 20 o C) for the high pressure processing is less effective than the treatment related to T = 4 o C and T = 50 o C. The decay of the number of microorganisms was particularly strong for the latter. It is also notable that the obtained decay of the changing their forms. The resulted denaturation curve extends in the pressure-temperature plane as shown in Figure 6. However, under high pressure appears additional: the break of cellular walls, the destruction of cellular organelle or changes in the permeability of cellular walls Starzonek et al. 2015). Moreover, the contribution of all these mechanism seems to depend on the distance from the denaturation curve in the P-T plane.
Concluding, it seems that the "revolutionary" expectations for totally safe human milk and with "native" nutritional, metabolic and immunological features, also during, the long term storage in a "home refrigerator" (~ 8 o C), are justified. However, this requires rather HPP+ treatment, than a simple implementation of the HPP technology used in the food industry.