Maxence Denavaut

Catherine Verret

Abdelhamid El Moueffak

Alain Largeteau

Delphine Quéveau

Marie M De Lamballerie

Abdelhamid El Moueffak

Effect of high pressure treatment on the characteristics of a model emulsion

Keywords: high pressure, physicochemical characteristics, microorganisms inactivation, emulsion

Introduction

Heat treatment may result in physicochemical modifications in the emulsions and in inactivation of bioactive compound properties making it difficult to preserve the microbial safety without using bactericidal compounds. [START_REF] Van De Ven | High pressure versus heat treatments for pasteurisation and sterilisation of model emulsions[END_REF][START_REF] Rigaldie | Sur l'impact des traitements sous hautes pressions dans la décontamination et la stérilisation de formes pharmaceutiques renfermant des molécules thérapeutiques sensibles aux procédés énergétiques[END_REF] Despite some slight modifications, [START_REF] Verret | Effects of high pressure on anhydrous milk fat crystallization in emulsion[END_REF] it has been shown that HP (high pressure) treatment has good results concerning the stability of both emulsions and bioactive compounds while permitting to inactivate different microorganism species. [START_REF] Van De Ven | High pressure versus heat treatments for pasteurisation and sterilisation of model emulsions[END_REF][START_REF] Rigaldie | Sur l'impact des traitements sous hautes pressions dans la décontamination et la stérilisation de formes pharmaceutiques renfermant des molécules thérapeutiques sensibles aux procédés énergétiques[END_REF][START_REF] Verret | Effects of high pressure on anhydrous milk fat crystallization in emulsion[END_REF][START_REF] Arora | Rheology and stability of acidified food emulsions treated with high pressure[END_REF][START_REF] Puppo | Effect of high-pressure treatment on emulsifying properties of soybean proteins[END_REF] This paper describes the effect of HP processing on the physicochemical characteristics of a non-food emulsion model and on the inactivation-recovery of various strains either inoculated separately or together in the product. The effects of the initial load and the storage duration have also been evaluated.

Materials and methods

The emulsion was prepared with a homogenizer under vacuum in a one-step process by addition of the aqueous phase into the oily phase under stirring. The two phases were first heated to about 70°C. Emulsions were cooled at room temperature to a constant stirring. The oil-in-water emulsion was composed of 21% of mixed vegetable saturated fatty acids and 79% of water containing 5% w/w ethoxylated alcohols as emulsifier agent. Samples consisting of 25 g of emulsion were vacuum-packed in a sealed plastic bag before being pressurized (500 MPa) for 10 min in a 3-L HP pilot unit (FRAMATOME, Paris, France; ACB, Nantes, France) at room temperature (20 ± 4°C). The level of pressure was reached at 3 MPa/s and then released almost instantaneously ( < 2 s).

Evaluated physicochemical characteristics were color measuring L*a*b* indexes using a spectrophotometer CM-3500d, MINOLTA, pH using a pH-meter, METTLER TOLEDO, particle sizes using a laser particle sizer Mastersizer S, MALVERN INSTRUMENTS LTD, and rheological behavior by flow method using a rheometer AR1000-N, TA INSTRUMENTS. Yield stress and apparent viscosity were calculated by modeling flow curves with Carreau-Yasuda equation using MATLAB software. Physical characterizations were checked 1, 90 and 180 days after HP processing, maintaining samples at 25°C during the storage.

Strains of interest were Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Aspergillus niger ATCC 16404 and Candida albicans ATCC 10231 (from MICROBIOLOGICS). Samples were inoculated with the strains either separately or together to obtain approximately 10 2 or 10 6 CFU/g of each. The number of surviving microorganisms was enumerated as colony-forming unit (CFU) in plate count agar after 24 h at 30°C and yeast glucose chloramphenicol after 72 h at 25°C for bacteria and fungi, respectively. Counting was done 1, 14 and 28 days after HP processing (up to 180 days for HP-treated samples), maintaining samples at room temperature during the storage.

Results and discussion

Effect of HP on physicochemical properties

Emulsion pH was between 4.2 and 4.3 and was not impacted either by HP treatment or by the storage duration. Table 1 shows the effect of HP over time up to six months on the different physicochemical characteristics followed. Pressurization of the emulsion at 500 MPa for 10 min did not lead to either modification of the particle size or rheological behavior. Observed differences between control and HP-treated samples concerning colorimetric indexes are very slight; moreover, control and treated samples evolve in the same way over time. It can be concluded that HP treatment has only few impacts on this emulsion until six months of storage at 25°C. These results are in accordance with those of Arora et al.and Puppo et al. [START_REF] Van De Ven | High pressure versus heat treatments for pasteurisation and sterilisation of model emulsions[END_REF][START_REF] Rigaldie | Sur l'impact des traitements sous hautes pressions dans la décontamination et la stérilisation de formes pharmaceutiques renfermant des molécules thérapeutiques sensibles aux procédés énergétiques[END_REF]

Effect of HP on microorganisms inoculated separately

Table 2 shows the effect of HP on the microorganisms inoculated separately over time up to 28 days for controls and up to 6 months for HP-treated samples.

The medium seems to have a lethal effect probably due to adaptation to the medium 1 day after inoculation. This effect occurs especially on P. aeruginosa, A. niger and C. albicans. Nevertheless, the fungi seemed progressively to recover or exceed their initial level 28 days after the inoculation. In the same period, bacteria appeared to be inactivated, probably due to the pH of the emulsion in association with other factors. HP (500 MPa, 10 min) at room temperature allowed a complete inactivation of the five strains with no recovery up to six months after HP processing. These results are obtained regardless of the initial load. a Values represent mean log 10 CFU/mL or CFU/g of unpressurized control samples and pressurized samples for 10 min (n = 3). b No CFU was detected in 1 mL of cell suspension from each of the samples tested.

Effect of HP on microorganisms inoculated together

Table 3 shows the effect of HP on the microorganisms inoculated together over time up to 28 days for controls and up to 6 months for HP-treated samples. When the five strains were inoculated together, the lethal effect observed the first day after inoculation was less pronounced especially for the low load in comparison with the strains inoculated separately. We could also observe a greater resistance of the bacteria to the bactericidal effect of the emulsion. As for the strains inoculated separately, the fungi were still present 28 days after the inoculation regardless of the initial load. When the five strains were inoculated together, HP also allowed a complete inactivation with no recovery until six months after HP processing. This result shows that the presence of several species did not induce pressure resistance properties.

Conclusion

The physicochemical characteristics of the emulsion were not impacted by HP processing. This kind of emulsion appeared to be a fungi selective medium. HP allowed a complete inactivation with no recovery until six months of the five microorganisms of interest inoculated separately or together directly in the medium, regardless of the initial load. The potential of HP processing to stabilize non-food medium has now to be confirmed on several other kinds of products. Moreover, it is also necessary to verify the innocuity of the treatment on bioactive compounds that could enter into medium compositions.

Table 1 .

 1 Effect of HP on the physicochemical properties according to storage time.

	Physical index	Storage time (day)	Control	500 MPa
	L *	1 90	81.4 ± 0.2 c 81.8 ± 0.1 c	81.5 ± 0.2 c 82.3 ± 0.1 d
		180	78.4 ± 0.2 a	78.8 ± 0.2 b
	a *	1 90	-0.70 ± 0.00 e -0.63 ± 0.01 g	-0.70 ± 0.00 e -0.65 ± 0.02 f
		180	-0.52 ± 0.02 i	-0.54 ± 0.01 h
	b *	1 90	-1.15 ± 0.05 k -1.04 ± 0.03 l	-1.25 ± 0.05 j -0.98 ± 0.06 m
		180	-0.05 ± 0.02 n	-0.07 ± 0.05 n
		1	2 . 9 ± 0.1 p	2.7 ± 0.1 op
	D 32 (µm)	90 180	2.7 ± 0.1 op 2.6 ± 0.1 op	2.6 ± 0.1 o 2.6 ± 0.3 o
		1	19.6 ± 1.2 r	16.7 ± 3.5 qr
	Yield stress (Pa)	90	16.7 ± 2.4 qr	16.0 ± 1.6 qr
		180	14.0 ± 0.1 q	13.5 ± 0.1 q
		1	59.9 ± 17.4 s	60.6 ± 4.9 s
	Apparent viscosity (Pa s)	90	68.8 ± 9.5 s	65.2 ± 7.5 s
		180	63.9 ± 3.6 s	58.8 ± 0.9 s

*

Note: Value with different superscript letters are significantly different (p < .05). * Values represent mean of unpressurized control samples and pressurized samples for 10 min (n = 4).

Table 2 .

 2 Effect of HP on microorganisms inoculated separately according to the storage time and the initial load. a

			Inoculum 10 2 CFU/g	Inoculum 10 6 CFU/g
	Microorganism	Storagetime (day)	Control	500 MPa	Control	500 MPa
	S. aureus	1	2 . 4 9 ± 0.04	ND	6.39 ± 0.12	ND
		14	< 1	N D	< 3	N D
		28	< 1	N D	N D	N D
		180		ND		ND
	E. coli	1	< 1	N D	5 . 8 2 ± 0.24	ND
		14	< 1	N D	< 1	N D
		28	ND b	ND	ND	ND
		180		ND		ND
	P. aeruginosa	1	< 1	N D	< 3	N D
		14	ND	ND	ND	ND
		28	ND	ND	ND	ND
		180		ND		ND
	A. niger	1	< 1	N D	3 . 6 9 ± 0.01	ND
		14	ND	ND	> 2.65	ND
		28	> 2.65	ND	> 2.65	ND
		180		ND		ND
	C. albicans	1	< 1	N D	4 . 5 2 ± 0.08	ND
		14	4.72 ± 0.01	ND	5.30 ± 0.03	ND
		28	4.70 ± 0.01	ND	6.08 ± 0.01	ND
		180		ND		ND
	Note: ND, not detected.

Table 3 .

 3 Effect of HP on microorganisms inoculated together according to the storage time and the initial load. a

			Inoculum 10 2 CFU/g	Inoculum 10 6 CFU/g
	Microorganism	Storagetime (day)	Control	500 MPa	Control	500 MPa
	S .aureus	1	3 . 7 9 ± 0.13	ND	5.99 ± 0.05	ND
		14	ND b	ND	1.64 ± 0.04	ND
		28	ND	ND	ND	ND
		180		ND		ND
	E. coli	1	1 . 9 1 ± 0.29	ND	5.72 ± 0.26	ND
		14	ND	ND	> 2.65	ND
		28	ND	ND	0.66 ± 0.26	ND
		180		ND		ND
	P. aeruginosa	1	< 1	N D	< 3	N D
		14	< 1	N D	> 2.65	ND
		28	ND	ND	ND	ND
		180		ND		ND
	A. niger	1	< 1	N D	3 . 3 9 ± 0.12	ND
		14	ND	ND	ND	ND
		28	> 2.18	ND	> 2.18	ND
		180		ND		ND
	C. albicans	1	1 . 3 1 ± 0.01	ND	4.49 ± 0.26	ND
		14	> 2.65	ND	> 2.65	ND
		28	> 2.65	ND	> 2.65	ND
		180		ND		ND

Note: ND, not detected. a Values represent mean log 10 CFU/mL or CFU/g of unpressurized control samples and pressurized samples for 10 min (n = 3).

b

No CFU was detected in 1 mL of cell suspension from each of the samples tested.