Natural Antimicrobials Combined with Ultrasound Treatments to Enhance Quality Parameters and Safety of Unpasteurized Strawberry Juice

Figures & data

Table 1. Box-Behnken experimental design matrix and mean values of all responses of untreated samples and samples treated under different experimental conditions after 14 d of storage at 5°C

	Independent variables
	Uncoded ^1			Coded			Experimental Responses ^2
Run	x1 US	x2 PE	x3 G	X1	X2	X3	YM	PSY	DPPH	TPC	Citric odor	Off-odor
1	0	180	0	-1	0	-1	8.17	6.65	67.17	141.66	0.10	3.57
2	0	180	0.3	-1	0	1	2.00	5.18	63.35	142.59	2.57	1.60
3	30	180	0	1	0	-1	8.29	6.81	68.24	147.24	0.23	4.43
4	30	180	0.3	1	0	1	2.65	2.70	55.79	133.73	1.93	1.33
5	15	0	0	0	-1	-1	8.26	6.55	65.28	138.18	0.23	4.10
6	15	360	0	0	1	-1	8.16	6.57	70.02	151.62	0.20	4.37
7	15	0	0.3	0	-1	1	2.65	2.70	53.51	99.45	1.93	1.53
8	15	360	0.3	0	1	1	2.65	2.70	63.24	142.60	2.00	1.33
9	0	0	0.15	-1	-1	0	3.00	4.36	58.25	135.72	1.30	1.53
10	30	0	0.15	1	-1	0	3.04	2.70	56.11	114.45	1.90	1.50
11	0	360	0.15	-1	1	0	3.10	4.98	66.20	135.99	2.17	1.23
12	30	360	0.15	1	1	0	2.85	2.70	64.28	177.45	2.07	1.80
13	15	180	0.15	0	0	0	3.50	2.70	68.20	130.50	1.77	1.07
14	15	180	0.15	0	0	0	3.15	2.70	64.39	137.63	1.83	1.17
15	15	180	0.15	0	0	0	2.95	2.70	66.67	142.10	2.27	1.57
Untreated sample (day 0)							4.37	4.23	60.93	99.90	0.00	0.00
Untreated sample (day 14)							8.13	6.65	53.05	130.31	0.57	4.37

¹Ultrasound time (US) is expressed in minutes; Pomegranate extract (PE) concentration is expressed in µg/mL; Geraniol concentration (G) is expressed in µL/mL;

²Yeasts and Molds (YM) and psyhrotrophic (PSY) counts are expressed in log CFU/mL; DPPH is expressed in % inhibition; Total polyphenols content (TPC) is expressed in mg GAE/100 mL; Citric odor and Off-odor are expressed in a 0–5 hedonic scale.

Table 2. Statistical parameters of RSM analysis of response variables

	Term^2
Response^1	Intercept	X1	X1^2	X2	X2^2	X3	X3^2	X1*X2	X1*X3	X2*X3
YM (adjR^2 = 0.9887; p <.001)
Coefficient	3.1910	0.0696	−0.1749	−0.0238	−0.0183	−2.8659	2.2594	−0.0730	0.1329	0.0255
SE	0.1520	0.0931	0.1371	0.0931	0.1371	0.0931	0.1371	0.1317	0.1317	0.1317
t-value	20.9869	0.7472	−1.2762	−0.2554	−0.1337	−30.7800	16.4855	−0.5547	1.0091	0.1940
p-value	<0.001***	0.4886	0.2579	0.8086	0.8989	<0.001***	<0.001***	0.6030	0.3593	0.8538
PSY (adjR^2 = 0.9391; p =.0012)
Coefficient	2.7000	−0.7820	0.8441	0.0795	0.1397	−1.6610	1.7896	−0.1548	−0.6606	−0.0041
SE	0.2512	0.1538	0.2264	0.1538	0.2264	0.1538	0.2264	0.2176	0.2176	0.2176
t-value	10.7481	−5.0836	3.7279	0.5165	0.6168	−10.7976	7.9032	−0.7114	−3.0367	−0.0191
p-value	<0.001***	0.0038**	0.0136*	0.6275	0.5644	<0.001***	<0.001***	0.5087	0.0289*	0.9855
DPPH (adjR^2 = 0.9300; p =.0017)
Coefficient	66.4171	−1.3191	−2.2906	3.8235	−2.9144	−4.3494	−0.4902	0.0535	−2.1569	1.2478
SE	0.7785	0.4767	0.7017	0.4767	0.7017	0.4767	0.7017	0.6742	0.6742	0.6742
t-value	85.3158	−2.7670	−3.2642	8.0204	−4.1533	−9.1235	−0.6986	0.0793	−3.1992	1.8508
p-value	<0.001***	0.0395*	0.0223*	<0.001***	0.0089**	<0.001***	0.5160	0.9399	0.0240*	0.1234
TPC (adjR^2 = 0.7929; p =.0229)
Coefficient	136.7453	2.1140	6.2516	14.9813	−2.0929	−7.5403	−1.6899	15.6810	−3.6100	7.4295
SE	4.4546	2.7279	4.0153	2.7279	4.0153	2.7279	4.0153	3.8578	3.8578	3.8578
t-value	30.6978	0.7750	1.5569	5.4920	−0.5212	−2.7642	−0.4209	4.0648	−0.9358	1.9259
p-value	<0.001***	0.4734	0.1802	0.0027**	0.6245	0.0396*	0.6913	0.0097**	0.3923	0.1121
Citric odor (adjR^2 = 0.8776; p =.0077)
Coefficient	1.9556	0.0000	0.0097	0.1333	−0.1069	0.9583	−0.7569	−0.1750	−0.1917	0.0250
SE	0.1738	0.1064	0.1566	0.1064	0.1566	0.1064	0.1566	0.1505	0.1505	0.1505
t-value	11.2534	0.0000	0.0621	1.2530	−0.6827	9.0057	−4.8324	−1.1628	−1.2736	0.1661
p-value	<0.001***	1.0000	0.9529	0.2656	0.5251	<0.001***	0.0047*	0.2974	0.2588	0.8746
Off-odor (adjR^2 = 0.9778; p <.001)
Coefficient	1.2667	0.1417	0.0750	0.0083	0.1750	−1.3333	1.3917	0.1500	−0.2833	−0.1167
SE	0.1085	0.0665	0.0978	0.0665	0.0978	0.0665	0.0978	0.0940	0.0940	0.0940
t-value	11.6716	2.1317	0.7667	0.1254	1.7889	−20.0628	14.2263	1.5960	−3.0146	−1.2413
p-value	<0.001***	0.0862	0.4779	0.9051	0.1336	<0.001***	<0.001***	0.1714	0.0296*	0.2695

¹Each response is informed with ANOVA parameters;

²Coefficients with p-value lower than 0.05 were retained in the models.

*Significant with p < 0.05; **Significant with p < 0.01; ***Significant with p < 0.001.

Figure 1. Response surface for microbiological quality of strawberry juice. Variation of yeast and molds (a) and psychrophilic bacteria (b) with ultrasound time and geraniol concentration at 180 μg/mL of pomegranate extract

Figure 2. Response surface curves for nutritional quality of strawberry juice. (a): Variation of DPPH with ultrasound time and pomegranate extract concentration at 0.15 uL/mL of geraniol. (b): Variation of DPPH with ultrasound time and geraniol concentration at 180 ug/mL of pomegranate extract. (c): Variation of TPC with ultrasound time and pomegranate extract concentration at 0.15 uL/mL of geraniol. (d): Variation of TPC with ultrasound time and geraniol concentration at 180 ug/mL of pomegranate extract

Figure 3. Response surface curves for sensory quality of strawberry juice. Variation of citric odor (a) and off-odor (b) with ultrasound time and geraniol concentration at 180 μg/mL of pomegranate extract

Figure 4. Response surface plots showing the combined effect of pomegranate extract concentration and ultrasound time (a), geraniol concentration and ultrasound time (b), and geraniol and pomegranate extract concentration (c) on the Desirability function with other variables constant at middle level

Table 3. Validation results

		Experimental Responses^1
Sample	Days of storage	YM (log CFU/mL)	PSY (log CFU/mL)	DPPH (% inhibition)	TPC (mg GAE/100mL)	Citric odor	Off-odor
Control	0	5.40 ±0.12	5.38 ±0.15	64.39 ±0.64	130.54 ±4.61	0.00 ±0.00	0.19 ±0.13
	14	8.33 ±0.11	8.30 ±0.12	58.20 ±2.24	276.71 ±6.61	0.00 ±0.00	4.65 ±0.12
Optimum treatment^2	14	4.69 ±0.11	4.60 ±0.10	74.45 ±2.78	313.45 ±23.29	2.07 ±0.20	2.13 ±0.26

¹Data is shown as means ± standard errors.

²Optimum treatment: 30 min of US, 360 μg/mL of pomegranate extract and 0.15 μL/mL of geraniol.

Table 4. Counts of inoculated Escherichia coli O157:H7 (log CFU/mL) in control and treated strawberry juice stored at 5°C

Sample	Storage time (days)
	0	3	7	11	14
Control	4.84 ±0.03^abA	4.59 ±0.04^aA	4.00 ±0.04^aB	3.47 ±0.07^aC	2.99 ±0.16^aD
Optimum treatment	4.65 ±0,05^bA	4.18 ±0.06^bB	3.22 ±0.04^bC	2.40 ±0.16^bD	< 2.00^bE

Optimum treatment: 30 min of US, 360 μg/mL of pomegranate extract and 0.15 μL/mL of geraniol.

Different lowercase letters indicate significant differences between treatments and different capitals indicate significant differences over time.