1. Introduction
Cleaning and disinfection are critical operations in dairy farming. On a commercial dairy farm, the clean-in-place (CIP) process is a standard process of the milking system, consisting of four cycles: (1) prewashing with warm water; (2) circular washing with alkaline chemical detergent; (3) circular washing with acidic chemical detergent; (4) sanitizing with sanitizing detergent before the next milking phase [
1]. The commercially used detergents in the CIP process typically contain sodium hydroxide, sulfuric acid, or phosphoric acid, which are highly caustic and can cause serious burns to the skin and mucous membrane [
2,
3,
4,
5]. Moreover, detergent residues in raw milk seriously affect the milk quality and reputation of the farms, which consequently lead to heavy economic losses to the farmers [
6].
Under such circumstances, research endeavors have been conducting to optimize the CIP protocols and find alternative detergents. Studies have shown that the optimization of cleaning protocols, aimed at minimizing the consumption of energy and chemical detergent, depends on the type of soils, surfaces, and materials to be cleaned [
7]. For the milking system, Vilar et al. [
8] found that weekly cleaning with acidic chemical detergent achieved a better hygienic environment than daily cleaning. Liu et al. [
9] suggested that using alkaline and acidic detergent alternately to clean the milk tank could help maintain a level of good hygiene and save water and costs. Overall, it is not suggested to apply alkaline and acidic chemical detergents together in one cleaning procedure, especially for equipment with clod surfaces, such as milking systems, because of the associated corrosivity, energy use, and complexity of the discharged water treatment afterward [
10]. Therefore, finding an alternative that is suitable for interchangeable use to replace these chemicals on sites would be highly advantageous to farmers.
Electrolyzed water (EW), as a novel cleaning and sanitizing agent, has been widely applied in food and medical industries [
11,
12]. EW can be disposed back into the environment after usage, contributing to decreasing the amount of halogenated compounds that accumulate in the environment, as highlighted by the U.S. Environmental Protection Agency [
13]. An EW generator with a semipermeable membrane can produce both alkaline electrolyzed water (pH of 11.5) and acidic electrolyzed water (AEW, pH of 2.6) at the cathode and anode in the same process, respectively [
14], and the characteristics of both solutions theoretically satisfy the requirements for alkaline and acidic chemical detergents in cleaning the milking system [
2]. Studies in the laboratory have shown that using alkaline electrolyzed water (pH of 11.5) and AEW (pH of 2.6) together in a complete CIP process could achieve an equivalent or even better cleaning effect [
2,
5,
15,
16,
17]. However, the practical challenge of using alkaline electrolyzed water and AEW together is the higher corrosivity. To optimize this protocol, Liu et al. [
18] showed that alkaline electrolyzed water as an alternative to alkaline chemical detergents could be used alone in standardized CIP processes for milking systems.
Meanwhile, an EW generator without a semipermeable membrane produced slightly acidic electrolyzed water (SAEW, pH of 5.0–6.5) in one study [
19]. Another study showed that AEW corrosivity decreased by more than 50%, with the pH increasing from 2.42 to 6.12 [
20], and the strong oxidability and good sterilization ability of the SAEW were also revealed [
21,
22,
23]. Additionally, the consumption of energy to produce SAEW is 50% less than that needed to produce the same quantity of AEW due to the different membrane structures. Theoretically, SAEW meets the standards for an acidic detergent alternative in the CIP process, however its effectiveness in cleaning milking systems has not yet been tested. The chemicals used in the CIP process are toxic, dangerous, and expensive, while using SAEW will help eliminate many of the dangers and will be cost-effective. The successful application of SAEW alone to wash milking systems would greatly benefit the optimization of the CIP cleaning protocols.
This work aimed to investigate the cleaning effect of using SAEW alone on three typical materials (stainless steel (304) pipes, rubber gaskets, and PVC milk hoses) that are often used in milking systems and to optimize the combinations of key parameters (treatment time, water temperature, and available chlorine concentration (ACC)) of the SAEW cleaning through response surface modeling.
2. Materials and Methods
2.1. Bacterial Cultures
This study selected
Escherichia coli (ATCC25922) and
Pseudomonas fluorescens (ATCC49642) as target bacteria, which can be easily found in raw milk. These two bacteria were collected from the China Veterinary Culture Collection (CVCC, Beijing, China and were cultured in 10 mL of tryptic soy broth (TSB, AOBOX Biotechnology Co. Ltd., Beijing, China) at 37 °C for 24 h. To ensure the culture broth had a bacterial population of 1 × 10
9 CFU/mL, each sample of the broth was checked using an aerobic plate count (APC) method with tryptic soy agar (TSA, AOBOX Biotechnology Co. Ltd., Beijing, China) [
15].
2.2. Milk Preparation
Raw milk was obtained from the Yulong dairy farm (Beijing, China). To increase the raw milk bacterial population, 1 mL of each culture broth was centrifuged at 4400×
g for 4 min at 4 °C [
15]. Two bacterial cells were resuspended together in 10 mL of raw milk with a mixer (WH-2, Shanghai Luxi Fenxiyiqi Co. Ltd., Shanghai, China).
2.3. Specimens Preparation
Three typical materials used in milking systems, namely stainless steel (304) pipes, rubber gaskets, and PVC milk hoses, were prepared as specimens. For each material there were 12 specimens, which were cut into 10 cm2 pieces. Before every treatment, the specimens were cleaned and autoclaved at 121 °C for 20 min.
To prepare contaminated specimens, 0.1 mL inoculated raw milk was evenly soiled on the whole surface of each specimen with a sterile glass-coated rod. Then, the specimens were dried using laminar flow for 2 h to evaporate all visible liquid. The initial concentrations of bacteria on stainless steel, rubber gasket, and PVC samples were 5.10–6.00 log10 CFU/cm2, 5.23–5.94 log10 CFU/cm2, and 3.00–5.64 log10 CFU/cm2, respectively.
2.4. Response Surface Design and Validation
A standard Box–Benken response surface design (
Table 1) was utilized to select suitable combinations of treatment time, water temperature, and ACC for SAEW cleaning, which contained 15 experimental trials, with the center point experiment repeated three times. According to the results of pre-experiments, the ranges for treatment time (8–12 min), water temperature (20–40 °C), and ACC (40–60 mg/L) for stainless steel pipe, and the ranges of treatment time (12–15 min), water temperature (20–30 °C), and ACC (40–60 mg/L) for rubber gasket and PVC milk hose samples were determined. For each trial three replications were performed.
An additional 6 trials with different parameters, which were not included in the model development, were carried out (
Table 2) to confirm the adequacy of the models.
2.5. Preparation of SAEW and Chemical Disinfectant
SAEW samples with different ACC levels of 20, 30, 40, 50, and 60 mg/L were generated by electrolyzing NaCl solutions using a generator (Beijing Rui’ande Environment Technology Co. Ltd., Beijing, China) set at 5, 8, 11, 14, and 17 A, respectively. The pH and oxidation–reduction potential (ORP) values of the SAEW were measured using a dual-scale meter (Hangzhou Ying’ao Technology Co., Hangzhou, China). The ACC was determined by a digital chlorine test kit (RC-2Z, Kasahara Chemical Instruments Co., Saitama, Japan). The chemical disinfectant was prepared at a concentration of 0.5% by diluting the concentrated acidic chemical detergent (Cidmax, DeLaval Co. Ltd., Tianjin, China; referred to as “super”) with tap water following the manufacturer’s instruction.
The SAEW and commercial acidic chemical detergent were heated in buckets with lids to the targeted temperature using a self-heating plate controlled by a single-channel thermal table (CH6E07, Beijing Kunlun Zhicheng Sensor Technology Co., Beijing, China).
2.6. Disinfectant Treatments
In order to check the cleaning potential when using SAEW alone, this study only performed the first and third steps of the standard CIP process (warm water rinsing and acidic chemical detergent washing) without disturbance from the final disinfection step. The cleaning process consisted of two cycles: (1) prewashing with 45 °C warm water for 5 min; and (2) washing with SAEW or “super”.
The cleaning solution was poured into a 400 mL beaker without any agitation to simulate the worst case scenario for locations of slow flow in the milking system [
15]. All prepared specimens were soaked in warm water, then transferred to the beakers containing SAEW or “super”. The treatment temperature and time for “super” were 80 °C and 8 min, respectively, according to the manufacturer’s recommendation. After treatment, specimens were taken out and sampled immediately.
2.7. Bacterial Counting and Cleanliness Evaluation
The soiling and hygiene levels of the specimens were evaluated before and after treatment with SAEW using the APC method and an adenosine triphosphate (ATP) bioluminescence test. The APC method is widely used to assess the cleanliness of food contact surfaces [
8]. The ATP bioluminescence test is well suited to monitoring the cleanliness within hazard analysis critical control point (HACCP) systems [
24]. It can be used for real-time assessment and can detect bacterial cells and food residues, which might affect the cleanliness and hygiene of food contact surfaces [
25].
Six specimens of each material were checked for their original contamination levels, while the other six specimens were checked for effects after cleaning. Three specimens were swabbed for microbiological analysis using sterilized cotton swabs soaked with 0.1% peptone water, while the other three were swabbed for ATP bioluminescence testing using the method recommended by the Ministry of Health, China [
26]. The details for the microbiology counting process were given by Liu et al. [
18].
As for the ATP bioluminescence test, samples were collected by using ATP test swabs (Shandong Langrun Commerce CO. Ltd., Jinan, China). Then, the swabs were tested using a LUMinator-T portable analyzer (CF-420, Shanghai Canfu Jidian Co. Ltd., Shanghai, China) to detect the emitted light from the ATP, which was quantified in “relative light units” (RLUs).
The bacteria and ATP removal rates were used for CIP performance comparisons. The equations used to calculate the bacteria and ATP removal rates were as follows:
where the units for the bacteria removal rate and bacterial concentration were % and log
10 CFU/cm
2, respectively.
where the units for the ATP removal rate and ATP value were % and RLU, respectively.
2.8. Statistical Analysis
The bacteria population was expressed as log10 CFU/cm2. The mean values for the total aerobic bacteria and RLUs were calculated from the independent triplicate trials. A Box–Benken response surface design table was generated and the results were analyzed using Minitab 17 (Minitab, Inc., State College, PA, USA). Significant differences in mean values for bacteria removal and ATP from three materials were analyzed using least significant differences with repeated measurement analyses of variance (ANOVAs) and a 95% confidence interval in SPSS 21.0 (SPSS, Inc., Chicago, IL, USA).
4. Conclusions
This study simulated a worst case scenario for the CIP process in a milking system, with low flow when rinsing with warm water and washing with acidic solution, in order to check the cleaning effects of SAEW on stainless steel, rubber gasket, and PVC hose samples, involving laboratory trials with appropriate combinations of treatment time, water temperature, and ACC. Compared to chemical detergent, the SAEW showed significantly higher bactericidal properties in conventional CIP steps, which suggested its potential for being a cleaning and bacteria removal agent for milking systems. For stainless steel, the cleaning SAEW parameters were optimized at 9.9 min (treatment time), 37.8 °C (temperature), and 60 mg/L (ACC); and were 14.4 min, 29.6 °C, and 60 mg/L for rubber gasket and PVC samples, respectively. The cleaning effect and stability of SAEW need to be verified in a real CIP system in the future.