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Ital J Food Saf
Ital J Food Saf
IJFS
Italian Journal of Food Safety
2239-7132
PAGEPress Publications, Pavia, Italy

10.4081/ijfs.2024.12217
Article
Low-frequency focused thermosonication for Salmonella typhimurium inactivation: an in vitro study
Lauteri Carlotta 1
Pennisi Luca 1
Di Clerico Daniele 2
Pennisi Viola 1
Vergara Alberto 1
Contributions: all the authors made a substantial intellectual contribution, read and approved the final version of the manuscript, and agreed to be accountable for all aspects of the work. 1 Department of Veterinary Medicine, School of Specialization in Inspection of Food of Animal Origin “G. Tiecco”, University of Teramo, Località Piano D’Accio;
2 Next Cooking Generation Srl, Milano, Italy
Department of Veterinary Medicine, School of Specialization in Inspection of Food of Animal Origin “G. Tiecco”, University of Teramo, Località Piano D’Accio, Italy. clauteri@unite.it
Conflict of interest: the authors declare no potential conflict of interest.

Ethics approval and consent to participate: not applicable.

Conference presentation: this paper was presented at the XXXII National Conference of the Italian Association of Veterinary Food Hygienists (AIVI), September 13-15, 2023, Maierato, Italy.

Publisher's note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Customer requests are addressed to safe products that best express their characteristics of “naturalness” and “freshness” for their entire shelf life; therefore, scientific research has been exploring the use of “non-thermal technologies”. Thermosonication using low-frequency focused ultrasound determines bacterial inactivation through the phenomenon of “cavitation”, guaranteeing high-quality standards of safety, nutrition, and freshness of the products. The present work aims to evaluate the effectiveness of the inactivation of Salmonella typhimurium in culture broth by low-frequency focused thermosonication with two different operational parameters: sublethal temperature (40°C, 50°C) and treatment time (5, 10, and 15 minutes). Treatment determined a bacterial load reduction compared to the negative control (untreated inoculum), which was statistically significant at the t-test (p<0.05). Average decreases of 1.5 log and 3.5 CFU/mL were observed, respectively, after treatment and after 24 hours of storage at +4°C. Treatment at 50°C for 15 minutes was the most effective (average value: 3.06 log CFU/mL; minimum value: 2.13 log CFU/mL; maximum value: 4.59 log CFU/mL). However, strains have shown markable variability: one of them even showed an increase in the microbial load 24 hours after treatment at 40°C for 5 minutes (-0.20 log CFU/mL); however, the same treatment showed a reduction of bacterial charge in all the other strains (average value: 1.05 log CFU/mL; minimum value: -0.20 log CFU/mL; maximum value: 2.28 log CFU/mL). This study poses numerous perspectives on the use of low-frequency focused thermosonication treatment in the food industry as a sustainable and safe alternative to classic thermal treatments.

Key words

Salmonella typhimurium
low frequency focused ultrasound
thermosonication
Salmonella inactivation in food matrix
Funding: none.
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pmcIntroduction

Every year, millions of people suffer from foodborne diseases; hence food safety is an imperative topic to hinder this trend (European Food Safety Authority and European Centre for Disease Prevention and Control, 2022). Traditionally, thermal treatments have been used to contrast microbiological hazards, but they can cause the loss of important compounds. Sanitization techniques called “non-thermal technologies” (i.e., ultrasound, hydrostatic high pressures, cold plasma, etc.) have allowed better retention of flavors, texture, color, and nutrient of foods than conventional thermal treatments (Beitia et al., 2023; Ferri et al., 2023). Among these techniques, high-intensity ultrasounds (<100 KHz) have attracted considerable interest because they are cheap, safe, “green friendly”, sustainable, and versatile (Lauteri et al., 2023). When ultrasounds propagate in liquid media, cavitation phenomena are generated (Huang et al., 2017). This phenomenon is the formation, growth, and collapse of microgas bubbles. Due to the pressure shocks up to several 100 MPa, the strong shock waves with 400 km/h microjets, and the production of hot spots at 5500°C, this occurrence improves microbial inactivation through severe damage to the cell wall (Suslick, 1988; Pokhrel et al., 2017).

Salmonellosis is the second foodborne pathogen in Europe, and poultry and swine food chains are the major pathways (European Food Safety Authority and European Centre for Disease Prevention and Control, 2022). It is pivotal that early elimination of pathogenic microflora from food hinders this trend. Ultrasounds employment as an alternative method of food decontamination is largely investigated in different food matrices: poultry skin (Musavian et al., 2014), meat and meat products (Wang et al., 2019), eggs (Bi et al., 2020), milk (Herceg et al., 2012) as well as in fruit and vegetable processing (Cruz-Cansino et al., 2016).

Research results suggest that the effectiveness of ultrasound technology is influenced by frequency, intensity, treatment time and treatment alone or combined with other technologies should be optimized for each food type. Indeed, the combined action of different technologies is more efficient than the use of ones allowed by the “hurdle technology method” (Lauteri et al., 2023).

The present study aimed to better understand the Salmonella typhimurium response against thermosonication treatment at different sublethal temperatures and times of treatment.

Materials and Methods

Bacterial strains

A total of 16 S. typhimurium strains, belonging to the biobank of the Unit of Food Inspection and Control of Animal Origin Food of the Department of Veterinary Medicine of the University of Teramo (Italy) were included in this study (Table 1). Inoculum was prepared in Mueller-Hinton broth (MH, Oxoid Thermo Fisher Scientific, Rodano, Milan, Italy) and incubated at 37°C for 18 hours until the early stationary phase. Cells were then harvested by centrifugation and washed three times with phosphate buffer saline (PBS) (Liofichem, Roseto degli Abruzzi, Teramo, Italy) 50 mM, pH 7.4. Inoculum was standardized to OD620nm0.1-0.2 (10×107 cells/mL) and then diluted to 10×106 cells/mL.

Test site

WavecoÒ (Next Cooking Generation, Milano, Italy), an ultrasonic bath with a capability of 30 L, was performed for focused ultrasound treatments. All samples were processed at 40 kHz with 100% amplitude with a focused ultrasonic wave according to the patent (international application No.: patent EP17733039.6). A special construction of the cover equipped with coils enabled the control and adjustment of the temperature of the medium.

Table 1. Salmonella typhimurium strains under this study.

Serovar	ID strain	Origin	
S. typhimurium	114	Meat product (sausage)	
S. typhimurium	115	Meat product (sausage)	
S. typhimurium	117	Meat product (sausage)	
S. typhimurium	118	Meat product (sausage)	
S. typhimurium	669	Cecal sample	
S. typhimurium	670	Pig carcass	
S. typhimurium	685	Pig carcass	
S. typhimurium	686	Pig carcass	
S. typhimurium	687	Cecal sample	
S. typhimurium	689	Slaughtering environments	
S. typhimurium	690	Slaughtering environments	
S. typhimurium	691	Slaughtering environments	
S. typhimurium	693	Pig carcass	
S. typhimurium	694	Pig carcass	
S. typhimurium	695	Pig carcass	
S. typhimurium	785	Slaughtering environments	

Table 2. Bactericidal effect of thermosonication treatments on Salmonella typhimurium.

	Descriptive value after treatments	Shapiro-Wilk	
	Mean*	SE	Median	SD	Min	Max	W	p	
Control	6.50	0.174	6.54	0.698	5.00	7.85	0.980	0.960	
40°C 5’ vitro	6.02	0.249	5.80	0.995	4.30	7.78	0.967	0.786	
40°C 10’ vitro	6.17	0.217	5.95	0.867	4.30	7.78	0.908	0.108	
40°C 15’ vitro	6.04	0.261	6.15	1.043	4.00	7.54	0.925	0.200	
50°C 5’ vitro	5.83	0.249	5.85	0.995	4.30	7.60	0.917	0.153	
50°C 10’ vitro	5.78	0.241	5.90	0.964	4.00	7.73	0.921	0.173	
50°C 15’ vitro	5.08	0.214	5.00	0.856	4.00	6.71	0.922	0.183	
SE, standard error; SD, standard deviation: *log CFU/mL.

Table 3. Bactericidal effect of thermosonication treatments on Salmonella typhimurium after 24 of storage.

	Descriptive value after treatments	Shapiro-Wilk	
	Mean*	SE	Median	SD	Min	Max	W	p	
Control vitro 24 h	6.71	0.175	6.69	0.700	5.24	8.09	0.971	0.850	
24 h 40°C 5’ vitro	5.66	0.260	5.38	1.038	3.91	7.53	0.960	0.670	
24 h 40°C 10’ vitro	5.20	0.215	4.95	0.859	3.30	6.86	0.900	0.082	
24 h 40°C 15’ vitro	5.30	0.216	5.33	0.864	3.00	6.54	0.920	0.171	
24 h 50°C 5’ vitro	4.71	0.252	4.78	1.010	3.10	6.54	0.926	0.211	
24 h 50°C 10’ vitro	4.47	0.238	4.68	0.953	2.70	6.43	0.920	0.168	
24 h 50°C 15’ vitro	3.65	0.205	3.65	0.819	2.50	5.21	0.937	0.318	
h, hours; SE, standard error; SD, standard deviation; *log CFU/mL.

Ultrasound treatment

The test involved a total of 16 S. typhimurium strains. More in detail, 9 mL of PBS (Liofichem, Roseto degli Abruzzi, Teramo, Italy) was added to 1 mL of 18-20 h S. typhimurium culture. Thermosonication antibacterial effect against S. typhimurium was performed by various ultrasound parameters: frequencies of 40 kHz, power of 800 W, time of 5, 10, and 15 minutes, and temperature of 40°C and 50°C. The temperature of the treatment medium was monitored during the whole process.

Microbiological analysis

Storage tests were conducted on all samples immediately after treatments and after storage, at 4°C for 24 hours by plating out onto xylose lysine deoxycholate (Liofichem, Roseto degli Abruzzi, Teramo, Italy) agar plates. One untreated sample (sample control) of every single strain was included in the analysis. Every strain was performed three times.

Table 4. Overview of Salmonella typhimurium response after treatments.

ID	C	SD	T1-1	SD	T1-2	SD	T1-3	SD	T2-1	SD	T2-2	SD	T2-3	SD	
114	6.64	0.23	6.46	0.14	5.95	0.45	6.53	0.71	6.63	0.53	6.15	0.79	6.18	0.63	
115	7.55	1.03	7.65	0.63	7.60	0.03	7.54	0.68	7.60	0.98	7.30	0.92	5.00	0.29	
117	6.52	0.59	5.30	0.85	5.78	0.89	5.60	0.49	5.85	0.53	6.00	0.92	4.30	0.56	
118	7.07	0.86	6.54	0.84	6.41	0.64	6.15	0.14	5.78	0.56	4.00	0.34	5.00	0.55	
669	6.74	1.01	5.70	0.10	5.70	0.90	4.00	0.63	5.85	0.32	5.78	0.45	4.00	0.33	
670	5.95	0.95	5.90	0.53	6.08	0.93	5.60	0.44	4.30	0.33	5.85	0.13	4.30	0.26	
685	6.56	0.19	5.30	0.89	6.15	0.54	6.15	0.81	5.78	0.55	6.23	0.12	5.60	0.18	
686	6.15	0.83	5.70	1.02	5.70	0.86	5.70	1.22	5.48	0.54	5.48	0.01	5.30	0.34	
687	5.78	0.02	5.00	0.40	5.95	0.23	6.30	0.51	4.30	0.82	6.04	0.98	4.30	0.71	
689	6.61	1.03	5.48	0.38	5.70	0.88	6.36	1.08	6.41	0.59	6.00	0.12	5.48	0.33	
690	7.10	0.59	6.98	0.89	7.27	0.34	7.20	0.72	5.90	0.33	5.00	0.54	6.71	0.29	
691	6.38	0.64	7.06	0.86	6.91	0.41	6.87	0.12	6.15	0.84	5.95	0.66	5.00	0.44	
693	6.18	0.01	6.23	1.21	5.90	0.36	5.60	0.77	6.04	0.37	5.60	0.59	5.30	0.31	
694	5.00	0.95	5.00	0.53	5,60	1.03	4.00	0.63	5.30	0.12	4.00	0.72	4.00	0.04	
695	7.85	0.19	7.78	0.89	7.78	0.81	7.54	1.09	7.60	0.32	7.73	0.72	6.43	0.81	
785	5.95	0.34	4.30	0.65	4.30	0.91	5.48	1.15	4.30	0.54	5.30	0.27	4.30	0.33	
ID, ID strains; C, control; SD, standard deviation; T1-1, 40°C - 5 minutes; T1-2, 40°C - 10 minutes; T1-3, 40°C - 15 minutes; T2-1, 50°C - 5 minutes; T2-2, 50°C - 10 minutes; T2-3, 50°C - 15 minutes.

Table 5. Overview of Salmonella typhimurium response after 24 hours from treatments.

ID	C	SD	T1-1	SD	T1-2	SD	T1-3	SD	T2-1	SD	T2-2	SD	T2-3	SD	
114	6.88	0.13	6.07	0.24	4.95	0.15	5.68	0.21	5.43	0.13	4.83	0.99	4.68	0.32	
115	7.79	0.23	7.23	0.34	6.40	0.25	6.54	0.18	6.45	0.28	5.87	0.12	3.20	0.73	
117	6.76	0.53	5.05	0.86	4.86	0.85	4.60	0.49	4.78	0.43	4.70	0.95	2.80	0.32	
118	7.31	0.36	6.30	0.39	5.50	0.44	5.15	0.54	4.71	0.56	2.70	0.35	3.50	0.88	
669	6.40	0.51	5.45	0.77	4.78	0.70	5.52	0.63	4.78	0.62	4.48	0.01	4.00	0.64	
670	6.19	0.92	4.75	0.63	5.04	0.53	5.30	0.44	3.23	0.43	4.74	0.10	2.80	0.32	
685	6.80	0.69	4.91	0.39	5.15	0.44	5.30	0.61	4.58	0.35	4.91	0.37	4.10	0.41	
686	6.39	0.93	5.31	1.72	4.70	0.82	4.85	1.52	4.28	0.74	4.16	0.21	3.80	0.77	
687	6.02	0.37	4.61	0.30	4.95	0.24	5.45	0.41	3.10	0.32	4.72	0.99	2.80	0.42	
689	6.85	0.03	5.23	0.18	4.78	0.77	5.36	1.38	5.35	0.29	4.70	0.25	3.98	0.33	
690	7.34	0.93	6.73	0.29	6.35	0.91	6.20	0.82	4.84	0.03	3.70	0.57	5.21	0.02	
691	6.62	0.64	6.82	0.86	5.99	0.01	5.87	0.22	5.08	0.74	4.66	0.85	3.50	0.74	
693	6.42	0.41	5.84	1.01	4.90	0.26	4.75	0.47	4.84	0.27	4.28	0.76	3.80	0.64	
694	5.24	0.82	4.75	0.63	4.68	1.03	3.00	0.43	4.23	0.22	2.70	0.66	2.50	0.01	
695	8.09	0.77	7.53	0.59	6.86	0.61	6.54	0.09	6.54	0.21	6.43	0.42	4.93	0.82	
785	6.19	0.31	3.91	0.35	3.30	0.13	4.63	0.15	3.10	0.51	3.98	0.31	2.80	0.21	
ID, ID strains; C, control; SD, standard deviation; T1-1, 40°C - 5 minutes; T1-2, 40°C - 10 minutes; T1-3, 40°C - 15 minutes; T2-1, 50°C - 5 minutes; T2-2, 50°C - 10 minutes; T2-3, 50°C - 15 minutes.

Statistical analysis

To evaluate the significant effect of ultrasound parameters, temperature, and treatment time on reducing the number of S. typhimurium, one-way analysis of variance (ANOVA) between subjects and t-test were applied with Tukey-HSD test at p<0.05 significance level. The normality of variance was tested by Shapiro-Wilk test; the homogeneity of variances was tested by the Levene test (XLSTAT 2014 software, Redmond, WA, United States) (Jamovi project, 2022).

Results and Discussion

Tables 2 and 3 showed the descriptive statistical results of S. typhimurium after treatment and after 24 hours after treatments.

In response to thermosonication treatments, the average bacterial count decreases, and this reduction becomes more pronounced with temperature increments and prolonged time, in accordance with Liu et al. (2021). Horwood et al. (1951) found that there was a relationship between initial bacterial count and ultrasound inactivation capacity; they noticed that there was a better efficiency of treatment when the bacterial load was low.

The t-test showed that the control group was significantly (p<0.05) distinguishable from the treated groups. The most effective treatment was at 50°C for 15 minutes in vitro essay, where the reduction was found to average 1.42 log after treatment and 3.96 log after 24 hours. ANOVA test showed that this treatment was significantly (p<0.05) distinguishable from the control group. The Tukey test supports the assumption that the applied ultrasound power and treatment duration have a significant effect on the decrease in Salmonella count compared to the control group.

Similar inhibition results were previously reported by Bi et al. (2020) and Liu et al. (2021): they found a 3.31 log reduction in S. typhimurium in liquid eggs after treatment at 968 W/cm2 and 35°C for 20 minutes and a decrease of 2 log of Salmonella in sprouts exposed to 200 W and 26 kHz for 5 minutes, respectively. It is important to underline that in the present study, PBS was used as a solution that is different from food matrices and may attenuate the ultrasound effect, as reported by Luo et al. (2022).

As shown in Tables 4 and 5, there was marked variability among different strains after treatment and after storage test.

Effectiveness evaluation showed that after the first treatment (40°C 5’), three strains (3/16, 18.75%) increased their bacterial count. The same increment was observed after treatments at 40°C 10’ (6 strains, 6/16, 37.50%), 40°C 15’ in (three strains, 3/16, 18.75%) and 50°C 5’ and 10’ (one strain, 1/16, 6.25%). In the evaluation of 24 hours post-treatment, only one strain showed an increment in bacterial count (40°C 5’). After 24 hours, there was better efficacy in the other treatments analyzed. Indeed, thermosonication technology causes wall damage to the bacterial cell, and after 24 hours, there was more sensible data than after treatments (Baumann et al., 2005; Pennisi et al., 2020).

Luo et al. (2022) studied ultrasound-resistant Salmonella and noticed that key enzymes of the tricarboxylic acid cycle were significantly downregulated, which led to a reduced adenosine triphosphate (ATP) content, although ATP activity was augmented. Salmonella tolerated ultrasound stress by upregulating their environmental sensing, chemiotaxis, substance uptake, and ATP production.

Conclusions

Ultrasound is an increasing field of research, particularly in the food industry, and it is effective against S. typhimurium. However, it is important to better understand the variability in response to treatment: bacteria could show adaptation to stress, like in antibiotic resistance (Ferri et al., 2022). It is also pivotal to better quantify the cell injury after treatment and evaluate the effectiveness of different Salmonella serovars. The ultrasound applicability combined with other methods for a synergic antimicrobial effect needs to be further investigated. To promote ultrasound application in the food industry, it is warranted to explore the interactions between acoustic energy and the food matrix.

Availability of data and materials

Data and materials are available from the corresponding author upon request.
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