


































Food Science and Nutrition Studies 

ISSN 2573-1661 (Print) ISSN 2573-167X (Online) 

Vol. 1, No. 2, 2017 

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71 
 

Comments on the High Pressure Preservation of Human Milk 

S. J. Rzoska1*, E. Rosiak1,2, M. Rutkowska1, A. Drozd-Rzoska1, A. Wesolowska3 & M. K. 

Borszewska-Kornacka4 

1 Institute of High Pressure Physics Polish Academy of Sciences, Warsaw, Poland 

2 Warsaw University of Life Sciences—SGGW, Warsaw, Poland 

3 Warsaw Medical University, Faculty of Health Science and Laboratory of Milk and Lactation 

Research at the Regional Bank of Human Milk, Warsaw, Poland 

4 Warsaw Medical University, Clinic of Neonatology and Intensive Care of the Newborn, Warsaw, 

Poland 

* S. J. Rzoska, E-mail: sylwester.rzoska@unipress.waw.pl 

 

Received: August 17, 2017    Accepted: August 26, 2017    Online Published: September 13, 2017 

doi:10.22158/fsns.v1n2p71        URL: http://dx.doi.org/10.22158/fsns.v1n2p71 

 

Abstract 

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(Bt2). 

Finally, issues regarding containers for the high pressure preservation of human milk are discussed. 

Keywords 

human milk, foods preservation, high pressures, thermodynamics, microbiology 

 

1. 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 = 



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62.5oC 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):  

 Microbiological safety even up to 90 days, 

 Taste, flavor and texture of fresh foods, 

 Vitamins content and nutritional characterizations of fresh foods, 

 Bioactivity close to the fresh product and long-time preserved, 

 The lack of chemical preservatives, 

 Using of salt can be avoided, 

 In selected cases deactivation of enzymes is possible, 

 The tool for the creation of new functional foods, 

 Used for already packed foods: the secondary microbiological contamination can be avoided, 

 Applicable for fluid and “solid” products, 

 Isotropic and “almost immediate” action of pressure on the product, 

 Environment-friendly method: no pollutions and it requires up to 10x less energy than the 

thermal pasteurization. 

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. 

 

2. 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 -20oC to 

120oC. 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 

Institute of High Pressure Physics Polish Academy of Sciences (Warsaw, Poland). Samples of human 

milk were collected via the Bank of Human Milk at Warsaw Medical University (see below). Prior to 



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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.  

 

3. Results 

3.1 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.  

 

Table 1. Selected Result Showing the Decrease of the Number of Key Parasitic Microorganisms on 

Compressing 

Microorganism n 

400 MPa 

n 

500 MPa 

n 

600 MPa 

T (
o
C) Ref. 

Staphylococcus aureus 5 log/30 min   21-31 [1] 

Staphylococcus aureus 6-8 log/30 min   21-31 [1] 

Staphylococcus aureus 5 log/15 min   4 [1] 

Staphylococcus aureus 8 log/15 min   50 [1] 

Staphylococcus aureus 2.5 log/15 min 5 log/15 min  4 [2] 

Staphylococcus aureus 2 log/15 min 4 log/15 min  20 [2] 

Staphylococcus aureus 3.5 log/50 min 8.5 log/15 min  50 [2] 

Streptococcus agalactiae 8 log/7 min   21-31 [1] 

Listeria monocytogenes 8 log/2 min   21-31 [1] 

Escherichia coli 8 log/10 min   21-31 [1] 

gram positive bacteria  5 log 5 log 30 [3] 

gram negative bacteria 5 log   30 [3] 

yeast, molds  300-400   30 [3] 

Note. The decay (cfu/mL) is presented in “customary n-log units” reflecting the change in the number of 

microorganism following the HPP process:  treateadnativen 10log . [1]—(Viazis, 2006; Viazis et al., 

2008), [2]—(Windyga et al., 2015), [3]—(Mimouni et al., 2017). 

 

http://pubs.acs.org/author/Vazquez-Landaverde%2C+Pedro+A
http://www.sciencedirect.com/science/article/pii/S0308814610006539


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Table 2. Key Metabolic and Immunological Factors of the Human Milk after the HPP Treatment 

and after the Reference “Classical” Thermal Holder Pasteurization 

Factor 0.1 MPa (Holder) 400 MPa/duration 600 MPa/duration T (
o
C) Ref. 

IgA 72% 100%/5 min 69.3% 12 [1] 

SIgA 51.2% 107%/30 min  30 [2] 

lysozome 51.2% 99.6%/6 min 72.7%/6 min 30 [2] 

α-Tocopherol 74.5% 71%/6 min 72.6%/6 min 10 [3] 

γ-Tocopherol 52.7% 83.3%/6 min 56.4%/6 min 10 [3] 

δ-Tocopherol 66.7% 102.9% 75%/6 min 10 [3] 

saturated fatty acids 99.3% 95.5%/6 min 98.8% 10 [3] 

polysaturated fatty acids 99.6% 99.2%/6 min 83.7%/6 min 10 [3] 

monounsaturated fatty acids 99.6% 90.8%/6 min 106.3%/6 min 10 [3] 

interleukin (6) 25.4% 35%/6 min 102%/6 min 10 [3] 

interferon 55% 93.8%/6 min 99% 10 [3] 

tumour necrosis factor 5% 100% 111%/6 min 10 [3] 

vitamin C 84% 100% 100% 20 [4-7] 

ascorbic acid 80% 100%/15 min 100% 20 [4-8] 

lysozome 56%  100%/15 min 20 [5-8] 

Note. Prepared basing on recent results from refs.: [1]—(Permanyer et al., 2010), [2]—(Viazis, 2008), 

[3]—(Delgado, 2014), [4]—(Moltó-Puigmartí et al., 2011), [5]—(Sousa et al., 2014), [6]—(Sousa et al., 

2016), [7]—(Mimouni et al., 2017), [8]—(Montserrat et al, 2016). 

 

Table 3. The Analysis of the Multi-Cycle HP Treatment (Duration t = 6-10 min) 

Factors Holder 

P = 0.1 MPa 

n% Temp. (
o
C)  number 

of cycles P = 325 MPa P = 425 MPa 

Nutritional: Energy 101% 101% 99.5% 4 1-4 

99% 99.5% 37 1-4 

Nutritional: Lipids 102% 98% 98% 4 1-4 

97% 97.6% 37 1-4 

Nutritional: Protein 102.2% 95% 99.2% 4 1-4 

92% 101% 37 1-4 

Immunological: IgA 66% 92% 94% 4 1-4 

92% 82% 37 1-4 

Immunological: IgG 68% 117% 90% 4 1-4 

112.5% 87% 37 1-4 

Immunological: IgM 32% 111% 73.5% 4 1-4 

104% 48% 37 1-4 



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Note. Prepared using results of ref.: (Girard et al., 2016). 

 

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. 

3.2 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 

compressing. The high pressure is applied for the time riset  up to the planned stationary value which 

is kept constant for the time .statt , usually between 3 and 15 minutes. Subsequently, the high pressure 

is released within decayt , down to ambient conditions. Pressure is transmitted to the product/sample 

via the pressurized medium, most often water with a possible addition of glycol or a similar liquid to 

facilitate the lubrication of elements of the high pressure processor and to avoid freezing during the 

Metabolic: lactoferrin 16% 100% 63% 4 1-4 

113% 84% 37 1-4 

Metabolic: lysozome 71% 97% 94.6% 4 1-4 

101% 99% 37 1-4 

Metabolic: lipase 48.5% 31% 43% 4 1-4 

70% 66% 37 1-4 

Bacterial load decay 

(“log units”) 

1.4-3.4  1.9-3.1 4 4 

 2.2-3.5 37 4 

~4.4  37 1 

~5.1  37 1+2 

~5.1  37 1+2+3 

~8 (?)  37 1+2+3+4 



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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 ( riset ) and decompressing ( decayt ) 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: .)(constCnRTPV  , 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). 

 

0 200 400 600 800 1000

0

100

200

300

400

500

600

P
re

s
s
u

re
  

(M
P

a
)

time  (s)

-10

-5

0

5

10

15

20

25

30

T
  

 (
o
C

)

 

Figure 1. The Form of the Pressure vs. Time Pulse Applied for HP Processing (Solid, Black Lines, 

Left Scale) 

 

Following this, for adiabatic heating/cooling associated with compressing/decompressing one obtains 

    1
1


 initialfinalinitialfinall PPTT , where Vp cc  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      12
1


 

initialfinalinitial PPRTfU .  



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0 200 400 600 800

0

100

200

300

400

500

600

P
re

s
s
u
re

 (
M

P
a

)

time  (s)

-10

0

10

20

30

T
  
(o

C
)

 

Figure 2. The Form of the Pressure vs. Time Pulse Applied for HPP Processing (Solid, Black 

Lines, Left Scale) 

 

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:  

    0,0,  PTdSPTS  and then   0, 
























 dP

P

S
dT

T

S
PTdS

PP

      (1) 

 
 

  
   




























P

P

P

T

P

T

S c
T

TSTT

PSVV

TS

PS

dP

dT





1

1
     (2) 

where P  is for thermal expansion coefficient, Pc  denotes the isobaric   is for density (Stanley, 

1971; Rzoska et al., 2010). 

The solution of the latter equation yields ca. KT 13  13 K temperature change for MPaP 400  = 400 MPa 

pressure change and KT 23  K for MPaP 600  = 600 MPa pressure jump, assuming water as the host 

pressurized medium. Notable is the similarity of the above dependence to the 2nd Ehrenfest equation 

(Stanley, 1971; Rzoska et al., 2010), used for describing the pressure evolution of the phase transition: 

  PP cTdPdT   , where P  and Pc  are for jumps of the expansion coefficient and the 

specific heat at the discontinuous phase transition. In the experimental practice for the HP technology 



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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 

VSdTdP   (Stanley, 1972; Rzoska et al., 2010). 

Figure 1 shows the most classical form of HPP pressure vs. time pulse. First, the pressure increases (for 

strise 125  125 s), subsequently it is kept at the processing pressure for .statt  (~ 5 min in Figure 1). 

Finally, pressure is released for stdecay 125  125 s. This is associated with heating above the base level 

up to KTrise 3.6  +6.3 K, returning to the base level during the high compression and finally, cooling 

below the base value by KTdecay 5.21  -8.1 K. Both values are below the maximal adiabatic 

heating/cooling values, what suggests the notable “isothermic contribution” during the rise and decay 

of pressure. Figure 1 is associated with the type I pressure processor, and the temperature measured on 

the axis of the pressure chamber, 5 cm. from its top.  

Figure 2 presents the pressure vs. time pulse, with the same value of strise 125  125 s and the sudden 

pressure decay down to the ambient value for stdecay 1.0  0.1 s. The rise of pressure is associated with 

KTrise 1.4  +4.1 K and the decompression related to KTdecay 5.21  -21.5 K. This result was obtained in type 

II pressure processor. It is visible that for the decay one approaches the adiabatic cooling temperature 

limit. This can be associated with the sudden, very short time decay of pressure. This scenario was 

realized by opening a valve linked to the pressure chamber. 

3.3 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). For fluids the container is most often a bottle, consisting from the main 

body and the closure cap with a sealing system. For the HPP technology the cap should have the same 

or higher compressibility than the main body. Consequently, the tightness of the closure can increase 

with rising pressure. A notable problem appears if filling of the bottle is not complete and the 

“parasitic” air-layer remains. Compressing water up to P = 600 MPa results in   %6.14%0  VV  

relative volume change. For P = 400 MPa one obtains   %5.11%0  VV . Air is ca. 8105.1   more 

compressible than water. All these may cause irreversible distortions or even break of the bottle when 

compressing. This issue is particularly important for HPP implementations in human milk banks, since 

the human milk is too precious to allow losses associated with the destruction of containers/bottles To 

solve the problem one can propose the design of the bottle given Figure 3a. It enables an additional 

anisotropic (longitudinal) shrinkage, what causes that even larger than 20% “parasitic”; air layer can be 

easily compensated.  



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cap
BA

bottlebottle

cap

 

Figure 3. The Proposal of Containers/Bottles for the High Pressure Preservation (HPP) of Human 

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. 

3.4 Microbiological Tests of the New HPP Protocol  

Figure 4 presents the decay of the number (N) of S. aureus bacteria in microbiologically contaminated 

human milk during such processing at P = 400 MPa for three selected isotherms. Such value of 

pressure seems to be close to the possible compromise between the antimicrobial safety and the 

preservation of key constituents (Tables I and II). For the applied semi-log scale the linear dependence 

is the indicator of the simple exponential decay    AtntN exp0 , i.e.,     AtntN  010 10ln1log  

of the number of microorganism for the compressing time t. This is the general advised pattern for the 

microbial deactivation under pressure (Balasubramaniam et al., 2016). A glimpse—test of Figure 4 

suggests that such pattern obeys for T = 4oC and T = 20oC isotherms, but definitively fails for T = 50oC. 

Figure 5 presents results of the distortions sensitive and derivative-based analysis of data from Figure 4. 



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In the basic case of the simple exponential decay one obtains:  

   AtntN exp0      AdttNd ln      (3) 

Such description is related to horizontal lines in Figure 5. However, it obeys only for T = 4oC and T = 

20oC isotherms, for the limited period of time. Results presented in Figure 4 indicate the occurance of 

the more general pattern:  

     2
0 expexp BtAtctC       BtAdttCd 2ln     (4)  

Horizontal lines in Fig. 5 indicate the clear “single” exponential decay    AttN exp . Solid curves 

are for the “extend” exponential decay      2expexp BtAttN  : (i) solid lines are for the process 

faster than the single exponential decay and described by B > 0, (ii) the dot-sloped line is for the 

process slower than the single exponential decay, described by B < 0 and (iii) for B = 0 the simple 

exponential case takes place. Results are obtained basing on experimental data from Figure 4. 

In eq. (4) the value B = 0 is for the simple exponential decay, B > 0 leads to the decay lesser that the 

simple exponential one and B < 0 is for the effective decay stronger the simple & basic exponential 

decay. The latter behavior is characteristic for the whole tested time range at T = 50oC. For T = 4oC and 

T = 20oC isotherms the notable change of the behavior during compressing takes place. It is visible that 

for the’ most convenient’ for applications isotherm T = 20oC the decay of the number of 

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  tstat. = 10 min, (ii) for  twait. = 10 min samples were kept at P = 

0.1 MPa, and finally (iii) the compression at P = 400 MPa for tstat. = 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. 

 



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0 5 10 15

10
5

10
6

10
7

10
8

10
9

7

 4 
 o
C

20
 o
C

50 
o
C

N
  
 (

cf
u
 /
 m

L
)

t  (min.)

lo
g

 [N
    (cfu

/m
L

)]

9

8

6

5

P = 400 MPa

 

Figure 4. Changes of the Amount of S. aureus in Human Milk during P = 400 MPa Compressing, 

for Selected Temperatures 

 

0 5 10 15

-1.0

-0.8

-0.6

-0.4

-0.2

 4 
 o
C

20
 o
C

50 
o
C

dl
nN

(t
 )

 / 
dt

t   (min.)

P = 400 MPa

 

Figure 5. The Derivative of the Amount (N) of S. aureus as the Function of Compressing Time 

 

The first moderate-pressure pulse (P = 200 MPa) did not influenced the total amount of bacteria but 

made them more sensitive to impact of the second “killing” high pressure pulse (P = 400 MPa). It is 

notable that the destruction of spores was also reported. One can speculate that the first pulse weakens 

the cohesion within cellular walls. Additionally, microbiological consequences of different forms of 

high pressure pulses shown in Figures 1 (type I) and 2 (type II) were tested. Finally, the storage studies, 

up to 14 days, were also carried out. For testing the impact of type I (Figure 1) and type II (Figure 2) 

advanced HPP+ technology human milk was contaminated by S. aureus up to the reference control 

level log 7.47 cfu/ml. The mix of three strains of Staphylococcus aureus: ATCC 25923 commonly used 

as indicator in high pressure inactivation tests; 4.4. strain isolated from food came from the Department 

of Industrial Food Microbiology University of Warmia and Mazury in Olsztyn; HM01 strain isolated 

from raw human milk in the Department of Food Hygiene Warsaw university of Life 

sciences—SGGW. 



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Table 4. Results of Microbiological Test When Applying Different Modes of HPP Treatments  

Note. pressure
day

native
dayDD nnn   : 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,  tstat. = 15 min at T = 20oC 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 9th 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 = 20oC. 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. 

 

 

Storage 

days 

Number of microorganisms during the storage 

Native 

Nn 10log  

n 

200+400 (II) MPa 

Relative values 

DDD nn   0/  

n 

200+400 (I) MPa 

Relative values 

DDD nn   0/  

0 7,47 4,88 2.59 3,09 4.38 

1 6,96 3,69 3.27/3.78 3,20 3.76/4.27 

2 7,17 4,10 3.07/3.37 3,75 3.42/3.72 

4 6,71 3,81 2.09/3.66 1,75 4.96/5.72 

9 4,43 1,79 2.64/5.60 0 4.43/7.47 

14 3,91 0  0  



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4. 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.  

 

 

Figure 6. The Sketch of the Denaturation Curve in the Pressure—Temperature Plane: Prepared 

Following Ref. (Smeller, 2002). 

 

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 = 20oC) for the high pressure processing is less 

effective than the treatment related to T = 4oC and T = 50oC. The decay of the number of 

microorganisms was particularly strong for the latter. It is also notable that the obtained decay of the 

number of microorganism is complex:      2
0 expexp BtAtntN  , i.e.,   2

10log BtAtCtNn  , 

where the constant 10lnlog 010 nC  . The distortions-sensitive analysis   BtAdttdn 2  revealed 

that the pattern and then the key mechanism of the decay of microorganisms can change after few 

minutes of compressing. It is worth recalling that for the thermal pasteurization the process is related 

mainly to the denaturation of proteins. One can link this to reaching the activation energy high enough 

to create new intermolecular links between secondary and ternary structures of proteins, definitively 



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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 (Rzoska et al., 2015; 

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” (~ 8oC), are justified. However, this requires rather HPP+ treatment, than a simple 

implementation of the HPP technology used in the food industry. 

 

Acknowledgements 

The authors would like to acknowledge the support of the grant “Lactotechnology as a response to the 

special nutritional needs of prematurely born children” (Natl. Centre for Res. & Develop. NCBiR, 

Poland: for years 2015-2017). ADR was supported by the National Science Centre (NCN, Poland) 

project, ref. 2016/21/B/ST3/02203.  

 

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