Application of Microbubbles Combining with Disinfectants to Inactivate Salmonella Typhimurium on Alfalfa Seeds and the Effects on Sprouting

Abstract

Microbial contamination is the main safety concern of sprouts and seeds are the major source. High concentrations of sanitizers (>10,000 mg/kg) are recommended for effective sanitation. Microbubble (MB) was reported to elevate sanitizer efficacy. Hence, MBs combined with disinfectants, chlorine dioxide (ClO₂, 500 ppm), and slightly acidic electrolyzed water (SAEW, 250 ppm), were used to inactivate Salmonella Typhimurium on alfalfa seeds. After fulfilling MBs for 10 min, alfalfa seeds were washed in 10 L of water for 10, 20, or 30 min. Compared with untreated seeds, S. Typhimurium reductions obtained by SAEW-MBs (SMBs) and ClO₂-MBs (CMBs) for 20 min were 3.8 and 3.3 log CFU/g, respectively. Conversely, the 20 min treatments of SAEW and ClO₂ only obtained reductions of 0.9 and 1.1 log CFU/g, respectively. More surface ruptures on the seeds treated with CMBs were observed under a scanning electron microscope compared with the ones treated by water and ClO₂ only. No adverse effects on the seed germination rate and the weight yield of sprouts were observed when treated with CMBs for 20 min. An MB device with capacity of 100 L was assembled and achieved reductions of 3.9 and 3.2 log CFU/g of natural microbes and S. Typhimurium, respectively, after 20 min CMB washing. Additionally, an MB device at 250 L was assembled and achieved 3.0 log CFU/g reduction in natural microbes. This study demonstrated that MBs enhanced the efficacy of disinfectants and could be applied in industrial-scale operations.

1. Introduction

Microbial contamination is the main risk to sprouts [1]. The most commonly consumed sprout is alfalfa sprouts, which are consequently involved in outbreaks associated with sprouts [2]. Salmonella serotypes are the most commonly occurring pathogens in those outbreaks. For example, S. Newport, S. Reading, S. Abony, S. Muenchen, and S. Kentucky in the USA [3,4]; S. Stanley in the US and Finland [5]; and S. Typhimurium and Havana in New Zealand [6] and Australia [7], respectively.

Contaminated seeds are the major source of pathogens in sprouts [6,8,9,10,11,12]. Furthermore, sprouting is operated in the conditions of a temperature of 22–24 °C and relative humidity of 95%, which is suitable for microbial growth [13,14,15]. Thus, disinfecting alfalfa seeds is a critical step for sprout safety. However, alfalfa seeds are difficult to disinfect due to their naturally rough surface [16]. Consequently, the US Food and Drug Administration (FDA) recommends 20,000 mg/L calcium hypochlorite (Ca(ClO)₂) to disinfect alfalfa seeds [17]. Due to this unusually high concentration, several alternative methods have been explored, including heating and other disinfectants, such as ozone (O₃) [18] and slightly acidic electrolyzed water (SAEW) [19,20]. Jaquette et al. [21] used 54 °C water for 5 min on seeds and obtained a 2 log CFU/g reduction in S. Stanley, but decreased the germination rate of seeds to 88%. In the same study, 1010 mg/L of NaClO was required to obtain a 2-log reduction. Yao et al. [22] treated seeds at 71.0 °C and 0.1 aw for 100 and 140 h to achieve 4.2 and 6.0 log reductions in Salmonella, respectively. Meanwhile, corresponding sprout yield ratios were 100.7% and 96.1%. In the same study, alfalfa seeds were treated with 20,000 mg/L NaClO for 15 min and 20 min and obtained 1.8 and 2.0 log reductions in Salmonella and sprout yield ratios of 70.9% and 65.1%, respectively. Another study applied SAEW (ACC 84 mg/L) for 3 h and obtained a 2.8 log CFU/g reduction in Salmonella [19]. Zhang et al. [20] used SAEW (25, 35, 45 mg/L) for 6 h on alfalfa seeds to obtain a > 1 log CFU/g reduction in Enterobacteriaceae bacteria.

Since the aforementioned single-step process required a long treatment time, combining methods for disinfecting seeds was also applied. More than 1 log CFU/g reductions in Salmonella and E. coli O157:H7 were achieved by combining O₃ water (5 mg/L, 20 min) and SAEW (ACC 10 mg/L, 15 min) for alfalfa disinfection when compared to O₃ water or SAEW only [23]. When alfalfa seeds were washed with 2% H₂O₂ for 10 min, vacuum-packed, then dry-heated at 73 °C for 4 h, a 7.1 log CFU/g reduction in S. Typhimurium was obtained. However, only a reduction of around 1 log was achieved when the seeds were treated with dry heat only [24].

Microbubbles (MBs) are defined by the International Organization for Standardization [25] as bubbles with a diameter smaller than 50 μm. MBs have been proven to have strong cleaning ability and can be produced in large volumes [26]. The diameter and density of MBs are closely related to the input air pressure, motor power, water quantity, and temperature [27]. Several studies have reported that the antibacterial abilities of disinfectants on food items were enhanced after being coupled with MBs [28,29,30,31,32,33,34]. Because MBs significantly increased the surface/volume ratio, retention time, and gas solubility in water, O₃ was the most commonly used disinfectant with MBs in the previous studies [28,30,32,33,35,36]. Alfalfa seeds without artificial inoculation were treated by water, MBs only, O₃ water (3.5 mg/L), OMBs (5.3 mg/L), and NaClO (5000 mg/L) for 5 min [32]. The highest reductions in natural microbes, 2.76 and 2.79 log CFU/g, were obtained from OMBs and NaClO, respectively. Additionally, germination rate and sprout weight were not affected by OMBs but were decreased by NaClO treatment. Though chlorine-based disinfectants are commonly used for fresh produce, combining MBs with chlorine-based disinfectants is seldom studied. MBs were combined with NaClO, ClO₂, or SAEW to treat S. Typhimurium and E. coli on sweet basil and Thai mint in 8 L of water [31]. After 5 min treatment with NaClO-MBs (NMB, 40 mg/L) and SAEW-MBs (SMB, 20 mg/L), 1.21–1.90 and 0.67–2.25 log CFU/g reductions in S. Typhimurium were obtained, respectively. During the following study, sweet basil and Thai mint were washed with surfactant (Tween 80, 1 g/L), then with SMBs (40 mg/L). Log reductions of more than 2 and 1 in S. Typhimurium and E. coli, respectively, were obtained when compared to MBs or SAEW only [36].

Among the chlorine-based disinfectants, ClO₂ and SAEW are commonly used. Therefore, MBs were combined with SAEW or ClO₂ to inactivate S. Typhimurium on alfalfa seeds in this study to achieve shorter treatment times and lower concentrations of disinfectants than previous studies. Physicochemical properties of the treated water, germination rate of seeds, and the weight yields of sprouts were determined. Additionally, the surface morphology of the treated alfalfa seeds with the inoculated S. Typhimurium was observed under a scanning electron microscope. In total, quantities of 10 L, 100 L, and 250 L of MB water were tested to achieve a basis for industrial-scale application. The theoretical basis of this study is that MBs detach the adhesive bacteria from the seed surface and suspend them in the surrounding disinfectant solution. This process is able to enhance the antibacterial efficacy and results in using lower concentrations of disinfectants and/or treatment time.

2. Materials and Methods

2.1. Microbubble Device

The injected atmospheric air and water were mixed in a Nikuni motor (KTM20ND07Z, Tokyo, Japan) with 750 watts (W) of power to generate MB water (Figure 1a). The air was injected at a flow rate of 2.5 L/min and the outflowing and inflowing water pressure was 4.5 and 2.719 kg/cm², respectively. The MB water flowed through a dissolution tank to separate large bubbles from microbubbles, and was then delivered into a beaker and circulated back to the motor. The water amount was maintained at 10 L. The MB fulfilling time in the beaker was 10 min, based on preliminary studies. For 100 L tests, a Calpeda motor (NGXM 6/18-60, Vicenza, Italy) with 1500 W power was used (Figure 1b), as in the previous study [37]. This system contained no dissolution tank and the filling time of bubbles was extended to 20 min. Air injection flow rate, outflowing, and inflowing water pressure were set at 1.0 L/min, 5.0, and 3.2/cm², respectively. The average diameters of the microbubbles of the Nikuni system and the Calpeda system were 29.05 ± 6.72 μm and 36.50 ± 5.91 μm, respectively [37]. Additionally, an industrial-scale MB device was assembled with a Calpeda motor (NGXM 6/18-60) and a 250 L stainless tank (Figure 2), in which two water inflow pipes were used. On inflow pipe produced a straight current to stir the seeds and the other produced tilted MB water inflow to generate a swirl current.

2.2. The Bacterial Culture and Inoculation of Alfalfa Seeds

S. Typhimurium (ATCC 13076, Bioresource Collection and Research Center, Hsinchu, Taiwan), was used as the testing bacterium. It was incubated in tryptic soy broth (TSB) at 37 °C for 18–20 h, then streaked onto a xylose lysine deoxycholate (XLD) plate to observe the colony morphology and uniformity. A well-isolated colony was inoculated into 10 mL TSB, then incubated for two consecutive 18–20 h incubations. After centrifuging at 5000× g at 4 °C for 5 min, the precipitate was re-suspended in 0.1% peptone water to 0.5–0.6 optical density at 600 nm (OD₆₀₀), which was at a level of 10⁸ CFU/mL according to the earlier study.

Alfalfa seeds were provided from Yiya Farm (Tainan, Taiwan) and examined based on the protocols of the Ministry of Health and Welfare (2013) to confirm no Salmonella contamination. Seventy g of alfalfa seeds were soaked in a flask that contained 100 mL of bacterial suspension. After shaking for 2 min, the bacterial suspension was discarded by pouring through a sterile stainless sieve. The inoculated seeds were collected on the sieve and placed evenly onto an aluminum foil in a laminar hood with the fan on for 14–16 h. Before testing, seeds were soaked in water for 1 h to imitate the common practice of the sprouting industry for facilitating germination. Preliminary tests confirmed Salmonella populations were around 5–6 log CFU/g after these procedures. All media were BD Difco™ (Franklin Lakes, NJ, USA).

2.3. Preparation of Disinfectants

ClO₂ (Emperor Chemical, Taipei, Taiwan) was freshly prepared before testing, based on the manufacturer’s instructions. SAEW was produced by electrolyzing sodium chloride solution (5 g/L) in a SAEW generator (Aquaox, Co. New Taipei, Taiwan). Several concentrations, 250, 500, and 750 mg/L of ClO₂ and SAEW, were tested during the preliminary study. However, the germination rates were reduced to around 50–60% at 750 mg/L of ClO₂ and at 500 and 750 mg/L of SAEW. Additionally, a <1 log CFU/g reduction in S. Typhimurium was obtained at 250 of mg/L of ClO₂. Thus, 500 and 250 mg/L of ClO₂ and SAEW were used, respectively, and pH was set at 6.0 for both disinfectants. The available chlorine (ACC) was measured according to the protocol of the Environmental Protection Administration, Taiwan (2020), in which available chlorine concentrations are determined by the titration of sodium thiosulfate (Na₂S₂O₃).

2.4. Testing of Microbubbles Combined with Disinfectants on Alfalfa Seeds

The treatments of ClO₂-MB (CMB) were conducted by using ClO₂ solution in an MB device based on our previous studies [29,35]. After 10 min MB fulfilling, 10 g of the inoculated alfalfa seeds were placed into the CMB water for 10, 20, or 30 min. The same procedures were applied for SAEW-MB (SMB) treatments, but only 20 min washing was used, since 30 min treatment damaged seeds and lowered the germination rate, and 10 min treatment was not adequate to inactivate bacteria based on the CMB results. Control groups included unwashed, water washing, MBs only, ClO₂ only, and SAEW only. The operational parameters were the same as in the combining tests. After washing, seeds were collected by pouring the water through a sterilized stainless sieve. The treated seeds were placed into a stomach bag with 90 mL of phosphate-buffered saline (PBS, pH 7.2), then stomached at 230 rpm for 3 min. After decimally serially diluting, 0.1 mL of the dilutant was spread onto plate count agar (PCA) and XLD agar plates, which were incubated at 37 °C for 18–24 h. To understand the remaining bacteria in the test water, 100 mL of treated water was collected after testing and filtered through a membrane (0.45 μm, cellulose nitrate, Sartorius, Göttingen, Germany). Two sets were tested, one membrane was placed onto a PCA, and the other was placed onto an XLD plate. Bacterial count was determined after incubation at 37 °C for 18–24 h. The same procedures were used for the 100 L volume test, except that 100 g of alfalfa seeds were used. No MB fulfilling time was used for the 250 L device, and non-inoculated seeds were used since they were tested in a commercial sprouting farm. After placing 1 Kg of seeds into the tank, the device was operated for 30 min. After washing, water was released from a pipe on the bottom, and the seeds were collected by a stainless-steel wire mesh strainer.

2.5. Measurement of Oxidation-Reduction Potential (ORP), pH, and Electrical Conductivity (EC) of Treated Water

Physicochemical properties, including temperatures, ORP, pH, and EC values, of the water from different treatments were measured. Water temperature was measured using an infrared thermometer at the end of treatment. Approximately 1 L of the treated water was sampled to determine ORP, pH, and EC values. The ORP values were measured using an ORP probe (ORP-148G, TECPEL, Taipei, Taiwan) connected to a pH/ORP meter (SP-2300, SUNTEX CO. New Taipei, Taiwan). The pH and EC values were measured using a pH/conductivity probe (serial 100 probe, Cole-Parmer, Vernon Hills, IL, USA) connected to a pH/conductivity meter (PC-200, Cole-Parmer, Vernon Hills, IL, USA).

2.6. Germinating Rate of Alfalfa Seeds and Weight Yield of Sprouts

After being treated with 10 L CMB for 20 min as previously described, 100 seeds were randomly selected and sprayed equally onto a germinating disk provided by YiYa Farm (Supplementary Figure S1). Controls were the unwashed and ClO₂ seeds. The procedures of seed germination and growth conditions were based on the protocol of Yiya Farm, which provided the seeds and germinating disks. Two disks were used for each treatment. The disks were placed in an enclosed chamber set at 22–25 °C and saturated humidity without illumination. Three disks were used for each treatment. In total, nine disks in three rows were used for each test. The disks of the same treatment were placed in different positions within each row. In the second test, the disks of the same treatment were placed in different positions from the first test (Supplementary Figure S2). The seeds were watered four times per day, and 1 L of tap water was used each time. The germination rates of seeds were determined daily for up to 4 days according to the formula: sprouting/100 seeds. Since sprouts are commercially harvested on day 7, the weight yield of sprouts was determined on day 7 according to the formula: the sprout weight (g)/seed weight (g)%. Meanwhile, characteristics of sprouts, such as appearance, texture, and aroma, were observed daily.

2.7. Scanning Electron Microscope (SEM) Observation

Twenty MB or CMB-treated alfalfa seeds with intact appearances were used for SEM observation. The control was the untreated seeds. Based on the methods [36], the seeds were soaked in a phosphate buffer containing 2.5% glutaraldehyde at 4–6 °C for 2 h. The seeds were dehydrated in serially increasing concentrations of ethanol solutions (50%, 70%, 80%, 90%, 99.5%), then frozen at −80 °C for 24 h. After lyophilization (Panchum Free Dryer FD-series, FD-8510T, Kaohsiung, Taiwan) for 24 h, the seeds were stored in a desiccator until fixed on a stainless stub and coated with gold-palladium (E1010, Hitachi, Tokyo, Japan). The morphology of the seeds was examined under a SEM (S3000N, Hitachi, Tokyo, Japan).

2.8. Statistical Analyses

Triplicate samples were tested for each test, which was conducted at least twice. The results were analyzed using the SPSS program (version 12.0, St. Armonk, NY, USA) and presented as average ± standard deviation. Significant differences (p = 0.05) between tests were determined by one-way ANOVA and Duncan’s test.

3. Results

3.1. Bactericidal Effects of MBs Combined with Disinfectants

Only 0.6, <0.1, 1.1, and 0.9 log reductions were obtained after 20 min washing with water, MB, ClO₂, and SAEW, respectively. However, the 20 min treatments of CMBs and SMBs achieved 3.2 and 3.8 log reductions, respectively (

Table 1. Populations (log CFU/g) and reduction (inside parentheses) of S. Typhimurium on alfalfa seeds after various treatments.

Treatments		CMB		SMB
Washing time	10 min	20 min	30 min	20 min
Untreated	5.50 ± 0.09 a	5.50 ± 0.09 a	5.50 ± 0.09 a	5.48 ± 0.18 a
Water soaking	5.13 ± 0.15 b (0.37)	5.13 ± 0.15 b (0.37)	5.13 ± 0.15 a (0.37)	5.13 ± 0.15 ab (0.37)
Water washing	5.08 ± 0.17 Ab (0.42)	4.93 ± 0.36 Aab (0.57)	4.50 ± 0.71 Ab (1.05)	5.01 ± 0.14 ab (0.47)
MB	5.21 ± 0.67 Aab (0.29)	5.48 ± 0.27 Aa (0.02)	5.32 ± 0.15 Aa (0.18)	5.46 ± 0.27 Ab (0.02)
ClO2	4.70 ± 0.23 Abc (0.80)	4.37 ± 0.45 Ab (1.13)	3.34 ± 0.14 Bc (2.16)	-
CMB	4.22 ± 0.21 Ac (1.28)	2.25 ± 0.27 Bc (3.25)	2.05 ± 0.30 Bd (3.45)	-
SAEW	-	-	-	4.60 ± 0.04 Ab (0.88)
SMB	-	-	-	1.65 ± 0.77 Bc (3.83)

Values represent means and standard deviations of six replicates (n = 6). MBs: microbubbles; CMBs: chlorine dioxide microbubbles; SAEW: slightly acidic electrolyzed water, SMBs: slightly acidic electrolyzed water microbubbles. Means with different capital letters in the same row are significantly different (p < 0.05). Means with different lowercase letters in the same column are significantly different (p < 0.05).

). Evidently, the combined treatments were more effective than disinfectants only (p < 0.05). A significant increase in the reduction in bacterial populations (p < 0.05) was obtained when washing time was increased from 10 to 20 min for the CMB treatment. However, extending washing to 30 min did not increase the reductions in bacterial population significantly (p ≥ 0.05), but caused obvious damage and lowered germination rates. Therefore, 20 min was considered the optimal washing time and used for the following treatments of SAEW and SMBs. A higher but not significant reduction was obtained from the SMB group when compared with the CMB group. However, the results of the SMBs were not as consistent as those of the CMBs and presented a larger standard deviation (Table 1). Thus, the treatment of CMBs for 20 min was used for the tests of large volume and sprouting. In the 100 L test, the natural microbes were reduced from 5.0 to 4.2, 3.3, and 1.1 log CFU/g (0.8, 1.7, and 3.9 log reduction) with water, ClO₂, and CMB treatments, respectively. The populations of S. Typhimurium were 5.2, 4.4, 3.7, and 1.9 log CFU/g for unwashed, water washing, ClO₂, and CMB, respectively. Corresponding reductions were 0.9, 1.5, and 3.2 log for water washing, ClO₂, and CMB, respectively. CMB treatment showed a significantly greater reduction (p < 0.05) than water and ClO₂ washing. Similar results were obtained from the 250 L tank; reductions in natural microbes were 0.8, 1.3, 3.0 log for water washing, ClO₂, and CMB, respectively.

Bacteria population was consistently undetected (detection limit = 1 CFU/100 mL) in the CMB-treated water, whereas 10–50 CFU/100 mL appeared in the ClO2-treated water for both the 10 L and 100 L tests. In contrast, approximately 2–3 log CFU/100 mL was obtained in the MB-treated water for the 10 L and 100 L tests. The results indicated that MBs alone did not possess strong antibacterial ability, but physically removed bacteria from the seed surface into the water. The main cause of the elevated antibacterial efficacy of combining MBs and disinfectants could be that MBs removed the adherent bacteria from the seed surface, and then disinfectants inactivated the free-suspended bacteria in the treated water.

3.2. The Physicochemical Properties of ORP, pH, and EC of the Treated Water

Since disinfectants were strong oxidants and adjusted to pH 6.0 before use; high EC and ORP values and slight acidity were observed. When combined with MBs, all EC, ORP, and pH values increased (

Table 2. Physicochemical properties of the washing solution of water, ClO₂, and CMBs after treating alfalfa seeds.

Treatments	Washing Time	pH	EC (μs/cm)	ORP (mV)	Temperature (°C)
Water washing	10 min	8.0 ± 0.0 a	342.3 ± 5.5 c	459.3 ± 14.8 b	25.2 ± 0.1 b
MB		8.2 ± 0.1 a	375.0 ± 18.1 c	433.3 ± 92.1 b	32.6 ± 0.2 a
ClO2		6.2 ± 0.0 c	764.3 ± 16.9 b	660.3 ± 4.6 a	25.1 ± 0.4 b
CMB		7.2 ± 0.1 b	848.7 ± 5.5 a	645.7 ± 8.6 a	34.7 ± 0.2 a
Water washing	20 min	7.9 ± 0.0 a	397.0 ± 76.7 c	441.3 ± 39.8 b	26.2 ± 1.3 b
MB		8.2 ± 0.0 a	384.3 ± 2.3 c	251.3 ± 54.3 c	37.2 ± 0.3 a
ClO2		6.1 ± 0.3 c	1175.0 ± 36.4 b	668.0 ± 9.5 a	26.4 ± 0.9 b
CMB		7.1 ± 0.2 b	1321.7 ± 37.6 a	651.8 ± 14.8 a	39.0 ± 1.6 a
Water washing	30 min	8.1 ± 0.0 a	405.0 ± 35.2 c	477.3 ± 29.7 c	26.0 ± 2.5 b
MB		8.4 ± 0.0 a	418.7 ± 9.1 c	272.3 ± 59.5 d	40.4 ± 0.9 a
ClO2		6.1 ± 0.1 c	1421.0 ± 29.6 b	664.7 ± 4.5 a	26.8 ± 0.1 b
CMB		7.2 ± 0.2 b	1697.5 ± 34.4 a	634.0 ± 14.6 b	43.5 ± 0.9 a
Water washing	20 min	8.0 ± 0.1 a	397.0 ± 76.7 b	411.3 ± 39.8 b	26.2 ± 1.3 b
MB		8.3 ± 0.1 a	410.7 ± 8.6 c	275.3 ± 35.5 d	36.4 ± 0.6 a
SAEW		6.1 ± 0.3 b	4026.7 ± 922.0 a	952.3 ± 23.0 a	26.6 ± 0.2 b
SMB		6.5 ± 0.4 b	4520.0 ± 739.0 a	942.7 ± 32.6 a	35.5 ± 2.2 a

The concentrations of chlorine dioxide and SAEW were 500 and 250 ppm, respectively. MBs: microbubbles; CMBs: chlorine dioxide microbubbles; SAEW: slightly acidic electrolyzed water; SMBs: slightly acidic electrolyzed water microbubbles. Values represent means and standard deviations of six replications (n = 6). Means in the same column with different letters are significantly different (p < 0.05).

). When water quantities increased to 100 L and 250 L, the trend of physicochemical characteristics was the same. Furthermore, the ranges of pH, EC, ORP, and temperature were very similar between different quantities.

3.3. Germinating Rate of Seeds and Weight Yield of Sprouts

On the first day, the germination rates ranged from 93 to 97%, and the highest rate (97%) was in the group of CMBs (Figure 3). Germination rates kept increasing and reached 99% on day 4, and no significant difference between treatments was shown throughout the 4-day observation. The weight yields of sprouts were 1209%, 1261%, and 1218% (p > 0.05) for water washing, ClO₂, and CMB treatments, respectively. Throughout the study, alfalfa sprouts grew well on the disks, and no differences in quality characteristics were observed between various treatments (Supplementary Figure S1).

3.4. Observation of SEM

The surface of alfalfa seeds is very uneven and provides many dents to harbor bacteria, which makes sanitation difficult. High numbers of bacteria covered large portions of the untreated seeds, but only few bacteria were observed on the MB- and CMB-treated seeds, particularly the CMB ones. These results demonstrated that MBs were effective in removing bacteria from seed surfaces. Under SEM observation, the surface of the MB- and CMB-treated seeds was obviously damaged, and evidently, the seed coat was broken. Furthermore, many debris were visible on the surface, particularly on the CMB-treated seeds. On the contrary, the surface of untreated seeds was intact, and almost no debris was seen (Figure 4). Since the first step of sprouting is the breaking of the seed coat, the cracks resulting from treatments did not interfere with seed sprouting, as shown in the seed germination results detailed in the previous section.

4. Discussion

4.1. Bactericidal Effects of Treatments

Disinfecting microorganisms on alfalfa seeds is a long-lasting and ongoing issue. Not only seeds could be contaminated with pathogenic bacteria, but bacteria multiply quickly during sprouting [9,10,11,12,36,37]. Our study also found that the total bacterial population increased from 1.7 log CFU/g on day 1 to 7.4 log CFU/g on day 3 during sprouting. Hence, this study investigated the antibacterial efficacy of various techniques, including the combination of disinfectants with MBs, disinfectants alone, MBs alone, and water, listed from most effective to least effective. These results clearly showed that coupling disinfectants with MBs was more effective in order to inactivate bacteria on the seed surface and in the treated water. These outcomes were consistent with previous studies [29,35,37] and further supported the concept that microbubbles and disinfectants possess a synergistic effect, in which the microbubbles diminish bacterial adhesion on the sample surface while the disinfectants inactivate bacteria in water. It has also been reported that bacteria adhering to surfaces or clustered together are more difficult to inactivate than those freely suspended in the disinfectant solution [38]. Thus, removing attached bacteria from sample surfaces was a critical step during the bacterial inactivation process of disinfectants. Furthermore, MBs generate free radicals, including OH^– and OH^•, during collapse, and also possess oxidizing capabilities, which could enhance antibacterial ability [30,39]. Furthermore, no bacteria in the CMB-treated water were detected. Thus, the probability of cross-contamination of seeds in the treated water was eliminated.

Another disinfectant, ozone (O₃), was also tested on alfalfa seeds by this research team, since combining O₃ with microbubbles (OMBs) has been reported to inactivate S. Typhimurium and E. coli on sweet basil leaves [34], leafy vegetables [40], tomatoes [29], eggs [37], and cabbage leaves [35]. While O₃ concentrations in water greatly increased via OMBs (from 2.45 to >10 mg/L at 30 min), O₃ and OMBs were deemed not to be very effective. With water washing as the baseline, only 0.39 log and 1.32 log reductions were obtained from 30 min washing using O₃ alone and OMBs, respectively. On the contrary, OMBs showed 4 and 5 log reductions on tomatoes [29] and eggs [37], respectively, using water washing as the baseline. This could be due to the rough surface of alfalfa seeds, contrasting with the smooth surface of tomatoes and eggs. For example, a reduction of around 2 log in S. Typhimurium and E. coli was obtained on cabbage leaves after OMB treatment [35], since the surface of cabbage leaves are less smooth than eggs and tomatoes but smoother than alfalfa seeds. Previous studies have also shown that applying ozone water to disinfect alfalfa seeds was ineffective. Singh et al. [18] applied O₃ water at 4.60, 9.27, and 14.3 mg/L for 3, 5, and 10 min against E. coli O157:H7 on alfalfa seeds. The maximum reduction was 0.54 log CFU/g. Mohammad et al. [23] extended washing time to 15 min with 5 mg/L O₃ but only obtained 0.5 and 0.6 log reduction in Salmonella serovars and hemorrhagic E. coli, respectively.

4.2. The Values of ORP, pH, and EC of the Treated Water

These results were similar to our previous studies [29,35,37]. The increase in EC and ORP values could be caused by the bursting of MBs, which generates free radicals. Additionally, the organic matter on food items washed and dissolved into the treated water could neutralize the acidity of disinfectants and increase pH values. However, all values of the treated waters were not significantly higher with longer washing time, except for the water’s temperatures. Higher water temperatures were shown during longer MB washing time, but not with ClO₂ and SAEW washing. This could be caused by MBs bursting, which has been reported to vibrate water molecules and increase water temperature [27].

4.3. Observation of Seed Surfaces Under SEM

SEM observations in this and previous studies [16,41] all showed the very tough surface of alfalfa seeds harboring bacteria. Thus, removing bacteria from the rough surface of alfalfa seeds is a critical procedure of disinfection. Another technique, ultrasound, which possesses strong cleaning ability, was also combined with ClO₂ to disinfect alfalfa seeds [1]. Higher but not significant reductions in E. coli and Salmonella Abony were achieved by combined treatment when compared with ultrasound only. This indicated that microbubbles possessed a higher removal ability than ultrasonication.

One of the main advantages of using microbubbles is quantity. The water volume of other techniques, such as ultrasound [1] and plasma-activated water [42], was less than 10 L. Additionally, previous MB studies tested volumes of 3, 4, 8 or 40 L [20,27,30,31]. In this study, we started at 10 L, then enlarged to 100 L, and eventually to 250 L.

5. Conclusions

In this study, significantly higher reductions in bacterial populations were achieved with CMB and SMB treatments than with ClO₂ and SAEW alone on alfalfa seeds. In addition, the treatment of CMBs for 20 min showed no negative effect on seed germination rate and sprout yield. SEM revealed that the rough surface of alfalfa seeds could harbor bacteria and microbubbles successfully detached those bacteria. Furthermore, microbubble devices with large quantities were presented to be effective and suitable for industrial use. However, the efficacy of this technique against other pathogenic bacteria and crops requires more studies to explore the antibacterial range.