A New High Hydrostatic Pressure Process to Assure the Microbial Safety of Human Milk While Preserving the Biological Activity of Its Main Components

Background: The main process used to pasteurize human milk is the low-temperature, long-time Holder method. More recently, the high-temperature, short-time method has been investigated. Both processes lead to the appropriate inactivation of vegetative bacterial forms but are ineffective against bacterial spores. Research Aims/Questions: We aimed to accomplish two main objectives: inactivation of all pathogens, including spores; and preservation of the activity of milk components. Design/Methods: Recently, a novel high-hydrostatic pressure process has been developed by HPBioTECH. Using the same raw human milk samples, we compared the effects of this method with those of the Holder method on vegetative and spore forms of pathogens and on bioactive components (lipase activity, immunoproteins). Results: Two main microbial strains were selected: Staphylococcus aureus (as a reference for vegetative forms) and Bacillus cereus (as a reference for spores). Use of the high-hydrostatic pressure process led to microbial decontamination of 6 log for both S. aureus and B. cereus. Additionally, the bioactivity of the main components of human milk was preserved, with activities of lipase, α-lactalbumin, casein, lysozyme, lactoferrin, and sIgA of ~80, 96–99, 98–100, 95–100, 93–97, and 63–64%, respectively. Conclusions: Use of this novel high-hydrostatic pressure process to generate microbiologically safe human milk may provide important benefits for preterm infants, including improved assimilation of human milk (leading increased weight gain) and improved resistance to infections. Because 10% of all human milk collected is contaminated by B. cereus, use of this method will also prevent waste.


INTRODUCTION
Human milk is the appropriate standard nutrient for infant development (1) and is also given to preterm and very low birth weight infants (2,3).
Two types of pathogens can contaminate such a medium: (i) endogenous pathogens from the mother and (ii) exogenous pathogens that mainly result from human milk collection by milk banks (4). Consequently, ensuring the safety and quality of donor human milk appears to be a crucial issue (5)(6)(7)(8).
Regarding microbial safety, both processes (LTLT and HTST) lead to appropriate inactivation of the vegetative forms of pathogens; however, these methods are completely ineffective against bacterial spores from exogenous contamination. During the last few years, two other human milk treatments have been developed: UV (14)(15)(16) and ultrasound (17,18).
In terms of preserving the activity of human milk after these pasteurization treatments, the LTLT process leads to many components (with nutritional enzymatic and immune properties) with reduced activity.
Over the last 25 years, high hydrostatic pressure (HHP) processes have been developed in food processing to mainly induce microbial safety (19)(20)(21)(22), which also consequently increases shelf life. Because HHP processes apply weaker energy than thermal ones, their main advantage is the preservation of the intrinsic properties of the treated medium. More recently, HHP processes have been extended to biological applications (23)(24)(25)(26)(27)(28)(29)(30).
The first industrial developments of HHP processes were established in Japan (1985)(1986)(1987)(1988)(1989)(1990). In this first approach (called a "conventional approach"), HHP processes were defined by only three main parameters: pressure (P), temperature (T), and duration of treatment (t). When HHP processes are managed by only these three parameters, high pressure values (450-600 MPa) must be applied to ensure high microbial safety (31), which has a negative consequence of inducing the modification of biological components or organoleptic properties of the handled product (32). If high temperatures (80-120 • C) are not used to induce detrimental modifications to biological activity, these HPP processes are ineffective at spore inactivation (33).

DEFINITION OF AN HHP PROCESS APPLIED TO HUMAN MILK
Different applications of this "conventional approach" to HHP treatment of human milk were tested with an emphasis on their ability to improve microbial safety. Viazis et al. (34) applied constant pressure (400 MPa) to human milk inoculated with different microorganisms [Staphylococcus aureus (ATCC 6538 and ATCC 25923), Streptococcus agalactiae (ATCC 12927), Listeria monocytogenes (ATCC 19115), and Escherichia coli (ATCC 25922)] to compare LTLT thermal pasteurization (Holder process) to high pressure treatment. The starting temperature was close to 21 • C to reach a temperature of ∼31 • C due to adiabatic compression heating. Six-to eight-log reductions were observed in microbial populations during treatment. Unfortunately, this HHP treatment used a conventional approach and was ineffective against bacterial spores, particularly Bacillus cereus spores, which represent a microbial strain observed in the contamination of fresh milk, heat-treated milk and human milk (35,36).

Research Aim
To establish an HHP process to inactivate both vegetative forms and bacterial spores contaminating human milk while preserving a substantial portion of the activity of milk components.

METHODS
Considering that high temperatures are rejected and that the pressure-temperature range required for spore inactivation would also lead to strong alterations of the biological activity of human milk components, an HHP process that could induce the germination of bacterial spores at lower pressure conditions (a moderate pressure value: P ≈ 350 MPa) was needed to preserve the biological activity of human milk as required for infant feeding.
Recently, a new approach to HHP processes was established by Demazeau et al. (37), and this approach accounts for parameters that characterize pressure delivery. Specifically, the compression rate (VA) or decompression rate (VD), application mode (MA) (continuous or cyclic) and latency time (t l ) between each cycle were defined.

Design
(i) To prove that this novel HHP was efficient for all pathogens with vegetative and spore forms, we performed a "challenge test." To validate this novel approach to HPP processes for the decontamination of human milk, we inoculated sterilized human milk at a level of 6 log with two main strains of microorganisms: S. aureus (ATCC 6538), which is a gram-positive vegetative bacterium resistant to pressure inactivation (38), and bacterial spores of B. cereus (ATCC 14579), a sporulated bacterial form that can induce severe intestinal infections (39). After applying various optimization tests, we defined the HHP experimental conditions based on 6 parameters that can inactivate all vegetative forms and bacterial spores (such as B. cereus spores).
The set of optimized process parameters was as follows: Pressure = 350 MPa, temperature = 38 • C, VA (application rate) = 1 MPa.s −1 , MA (application mode) with n a (number of cycles) = 4 cycles and t a (duration of each cycle) = 5 min, and t l (latency time with normal pressure between each cycle) = 5 min.
(ii) To demonstrate the conservation of bioactive components of human milk, we compared raw human milk with pasteurized and HHP treatments of the same sample. We measured the main biologic components of human milk, including lipase activity, lactoferrin, lysozyme, and IgA under either Holder pasteurization (62.5 • C, 30 min) or novel high hydrostatic pressure.

Setting
The high-hydrostatic pressure machine is located at HPbioTech, which is situated 10 km from the Human Milk Bank (HMB) of Bordeaux-Marmande.
We used human milk after consent from the mother. Raw human milk is pasteurized and stored at −18 • C at HMB; when transferred to HPbioTech, it is stored at −80 • C until analysis.

Sample
The pasteurized human milk was used to set of optimized process parameters of HHP. Sterile human milk was inoculated with 5-6 log S. aureus or B. cereus and treated with the optimized set of HHP. This was termed the "Challenge test." The raw human milk was treated with the optimized set of HHP to measure the biologic products of human milk, such as lipase activity and immune proteins lactoferrin, lysozyme, IgAs.
After HHP treatment, the challenge was employed to identify conditions that allow for destroying vegetative and spore forms of bacteria and preserving lipase activity and immune bioactive proteins.
Compared to Holder pasteurization, which destroyed 0 spores (see Table 1), the HHP sample size was determined to be 6 log of B. cereus spores and 6 log of S. aureus. The value obtained after Holder pasteurization showed no destruction of B. cereus, but no B. cereus was found after HHP treatment. Thus, very few samples are needed (see Table 1). We measured the reproducibility of destroying 6 log B. cereus in 3 repeated HHP treatments.
The Holder treatment destroyed all lipase activity (activity = 0), whereas between 70 and 100% of residual activity was found with the HHP treatment (see Table 3).

Data Analysis
We first verified the normality of the population and the homoscedasticity of variances. If verification was achieved, we used the Student t test to compare the two treatments (Holder vs. HHP); if not, we used the non-parametric test.
Two tailed p < 0.05 indicated significance.

Ethical Consideration
The milk used in this study was derived from the Human Milk Bank of Bordeaux-Marmande. Prior to donating milk, each mother signed a consent form indicating that any discarded milk could be used for research purposes. We therefore did not require approval for this study from the local Ethics Committee. Moreover we can utilize human milk samples that cannot be used because the mother smokes or there are other contraindications to its donation.   (Figure 1). Bioactive, antimicrobial and immune proteins: lactoferrin, lysozyme and IgA, showing more or less broad ranges of functions, were analyzed using Enzyme Linked ImmunoSorbent Assays (ELISAs) based on the sandwich technique; the antibody directed against the protein to analyze is pre-coated on the surface of microtiter wells. A biotinylated detection antibody is then added to the wells to bind to the captured protein. Streptavidin-conjugated horseradish peroxidase (SA-HRP) is then added to catalyze a colorimetric reaction with the chromogenic substrate 3,3 ′ ,5,5 ′ -tetramethylbenzidine. The colorimetric reaction produces a blue product, which turns yellow when the reaction is terminated by addition of dilute sulfuric acid. The absorbance of the yellow product at 450 nm is proportional to the amount of protein present in the sample. The protein concentrations in the test samples can then be quantified by interpolating their absorbance from the standard curve generated in parallel with the samples (41).

Lipids
Lipase activity (Institut Biochimie Nutrition ITERG, Dr. C. Vaysse-Dr. L. Couedelo). The lipase activity compared to the substrate was monitored by quantitative release of fatty acids and glycerol generated during the hydrolysis of TAG lipase. The hydrolysis reaction is automatically followed by pH measurements via neutralization of fatty acids liberated by the enzyme over time by a standard solution of sodium hydroxide (NaOH 25 mmol/L) at pH 8 ± 0.2.
The results are expressed as the in International Units as micromoles of fatty acids liberated per minute and per milliliter of breast milk.
We used the granulometry of lipid droplets of human milk: raw, pasteurized, and novel HHP of the same batch (Figure 2).

Microbial Spore and Vegetative Destruction
In these experimental conditions, total inactivation of the microbial contamination of human milk was possible in challenge tests with S. aureus and B. cereus spores.
The IE of the new HHP process for human milk inoculated with spores of B. cereus (ATCC 14579) and an evaluation of its reproducibility (n • 1, n • 2, and n • 3) are provided in Table 1. N i and N HHP are, respectively, the initial (prior to HHP treatment) and final (after HHP treatment) microbial concentrations. IE is provided for HHP treatment Tables 1, 2 provide the corresponding inactivation efficiency (IE) of each microorganism. The HHP experiment and microbial analysis were repeated 3 times (n • 1, n • 2, and n • 3) for the same i.e., 1.5E+06 Colonies Inactivation Efficiency (IE) = 6.18 Log In addition, this HHP process was evaluated for the inactivation of cytomegalovirus (CMV). This virus was selected due to its risk of human milk contamination and risk of postnatal infection (40,(42)(43)(44).
Different attempts were made using a suspension of cytomegalovirus with an initial viral particle concentration of up to 7 log. After application of the HHP treatment, which was characterized by the optimized process parameters used for the inactivation study of either S. aureus or bacterial spores of B. cereus, total CMV inactivation was observed.

Activity Retention of the Main Constituents of Human Milk
As a first evaluation, three main types of human milk components were selected: -A component with enzymatic properties (lipase), -Components with antimicrobial properties (lysozyme and lactoferrin) or with immunological properties (IgA), and -Components with nutritional properties.

Impact of the HHP Process on Lipase Activity
An evaluation of lipase activity was important to compare the biological effects of this HPP process with those of thermal LTLT treatment (induction of total inactivation). The experimental conditions of HHP treatment were the same as those used for microbial decontamination (P = 350 MPa, T = 38 • C, VA = 1 MPa.s −1 , MA = 4 cycles of 5 min and t l ± 5 min).
Residual activities of the lipase enzyme in three samples (raw milk, LTLT-pasteurized milk and HHP-treated milk) and in three replicates (n • 1, n • 2, and n • 3) for HHP treatment are provided in Table 3. Due to the variability of lipase activity in human milk, Frontiers in Public Health | www.frontiersin.org  Table 3).
The residual activity of the lipase enzyme after HHP processing of human milk was between 80 and 85% of the initial value of human milk treated at 38 • C.
A comparison with the residual activity from the conventional HHP process [reproducing Viazis's et al. (34) HHP treatment] resulted in a residual lipase activity of close to 75%.

Impact of the HHP Process on the Activity of Different Components With Antibacterial or Immune Properties
The activity of biological components (lysozyme, lactoferrin, αlactalbumin, and IgA) was also evaluated before and after HHP processing of human milk using the same set of experimental parameters (Table 5; Figure 1).
Considering the differences between our HHP process and those described in the literature for human milk, the following remarks can be made.

Impact of the HHP Process on Human Milk Components With Nutritional Properties Milk Fat Globule (MFG) Granulometry in Raw, LTLT-Pasteurized, and HHP-Treated Human Milk Using This Novel HHP Process
Evaluation of the size of MFGs showed that the population was bimodal with an approximately equivalent average diameter (d4.3) for all types of milk (raw milk: 5.5 µm; LTLT: 5.6 µm; and HHP: 5.4 µm). In addition, the proportion of "small MFGs" was greater in raw milk and HHP-treated milk (d3.2 = 0.6 vs. 0.8 µm, respectively) compared to that in LTLT-pasteurized human milk (d3.2 = 3.1 µm). This result suggests that the size structure of raw breast milk is preserved by HHP treatment, whereas LTLT promotes coalescence and therefore increases the (A, B, and C correspond to 3 different human milk samples, and n • 1, n • 2, and n • 3 correspond to HHP treatment reproducibility assays. Lipase activity of raw human milk samples and milk samples after thermal LTLT pasteurization and HHP processing (A, B, and C correspond to 3 different human milk samples, and n • 1, n • 2, and n • 3 correspond to HHP treatment reproducibility assays) The lipase activity was completely destroyed (0% lipase activity) by LTLT, whereas lipase activity after the new HHP remained similar to its original value (Raw Human Milk) between 78.6 and 100% with a mean of 87.8% (Wilcoxon test p = 0.25).
number of "large" MFGs. In addition, the total fat content was similar regardless of the performed treatment (raw, LTLT and HHP: 34.0, 34.1, and 32.3 mg/mL milk, respectively) and of the fatty acid profile of the milk.

DISCUSSION
For the first time, this new HHP process for the microbial safety of human milk can irreversibly inactivate both the vegetative forms of microorganisms, such as gram-positive bacteria including S. aureus, and bacterial spores, such as those of the contaminant B. cereus, while preserving at least 80% of the biological activity of the main components. Previously reported works involving HHP treatments were based on socalled "conventional" approaches in which the applied pressure was not controlled (15). Table 4 provides average values of the resulting microbial safety using three processes (LTLT, HTST and this new HHP) on human milk with S. aureus (grampositive bacterium) and B. cereus (sporulated bacterium) as contamination references. Consequently, inactivation of bacterial spores, such as those of B. cereus, was not possible with a technique other than the new HHP.
The retention rates of the biological activity for different components with specific properties [BSSL lipase (with enzymatic properties), lysozyme and lactoferrin (characterized by antimicrobial properties), and IgA (with immunological properties)] are summarized in Table 5.  Comparisons with the HTST process suggest that the retention rates of the biological activity of human milk components vary widely (particularly for BSSL) by author (9)(10)(11)(12)(13). In a recent paper by Giribaldi et al. (45), two aspects of the impact of the HTST process were evaluated: (i) microbial inactivation but not destruction of B. cereus and microbial spores and (ii) retention of the biological activity of human milk components using a specific HTST device for human milk pasteurization. Residual lysozyme activity was between 95% and 100% after application of our HHP process. Our value agrees with that reported by Viazis et al. (46) (96%) following HHP treatment of human milk at 400 MPa and 20 • C. Viazis's et al. (34) HHP treatment resulted in a residual lipase activity of close to 75%.
Mayayo et al. (47) found that treatment at 300, 400, 500, and 600 MPa for 15 min and T = 20 • C using the "conventional approach" to HHP processes denatured 9, 23, 34, and 48% of lactoferrin, respectively. In our approach, the retention rate of lactoferrin was over 93% (denaturation was below 7%) despite using a temperature of 38 • C to limit the germination of B. cereus spores.
The residual activity of IgA was comparable to that obtained by Delgado et al. (48) (47.5% at 300 MPa and 50 • C). In an early paper, Viazis et al. (46) found that high-pressure processing of human milk using the "conventional approach" at 400 MPa for 30, 60, 90, and 120 min and at a treatment temperature close to 31 • C resulted in 85.6, 87.1, 80.6, and 75.4% retention, respectively. Permanyer et al. (49) claimed that after a treatment at 400 MPa for 5 min at 12 • C, 100% of IgA activity was maintained, whereas IgA retention was 87.9 and 69.3% at higher pressure conditions (500 and 600 MPa, respectively). Contador et al. (50) evaluated the retention activity of IgA after highpressure treatment at different pressures (400 and 600 MPa) and different treatment durations (3 and 6 min) with an initial temperature of 10 • C at 400 MPa for 6 min; the retention of IgA activity was close to 90%.
Comparisons of these research studies suggest that IgA activity mainly depends on both the pressure and temperature of highpressure treatment.
The retention rates of the biological activity for different components with specific properties [BSSL lipase (with enzymatic properties), lysozyme and lactoferrin (characterized by antimicrobial properties), and IgA (with immunological properties)] are summarized in Table 5.

LIMITATIONS
The new HHP process requires 90 min to treat human milk vs. 60 min for the Holder method; however, the cost of the HHP device is more than is a conventional pasteurizer. However, the new HHP saves up to 10% of material contaminated by B. cereus. For example, the Bordeaux Human Milk Bank collects 11,000 liters of human milk per year; 10% of this amount (or 1,100 liters) is contaminated with B. cereus and therefore must be discarded (51). This represents 165000e /year, lost with the conventional pasteurizer per year, which would not be rejected with the new HHP.

CONCLUSION
This new HHP process is promising for implementation in human milk banks based on a comparison of three processes for the microbial safety and retention of the biological activity of different milk components. This approach is the first process that can inactivate bacterial spores, such as those of B. cereus; this point is important due to the risks of bacterial spores to preterm or young infants (52).

AUTHOR CONTRIBUTIONS
GD and AP processed the human milk samples, and the results of bacteriological analyses were verified in double-blind experiments performed by PL of CHU. GD worked with AP to write the first version of the manuscript; unfortunately, GD is now deceased. PL performed a double-blind bacteriological study on the HHP samples from GD at the CHU. LC and PM performed the experiments, analyzed the data and wrote their part of manuscript and participated in revising the manuscript. Technis and results of Bacteriology was revised by PL and AP. The technics and results of Lipids by LC. The technics and results of proteins by PM. CB wrote the manuscript.

FUNDING
We received a grant of 150.000e from the Conseil Regional d'Aquitaine.