Microbiological Evaluation of Hot Filling Production Line

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After more than ten years of development, domestic beverage hot filling has formed the pattern of common hot filling, ultra-clean hot filling (a transitional model between the common hot filling and aseptic filling) and aseptic filling. For many small and medium-sized enterprises, due to the overall design problems, batch quality problems will easily occur in the production process if the management is improper. However, the aseptic production line has a high production cost and large capital investment. Therefore, the ultra-clean hot filling production line between the ordinary hot filling and aseptic cold filling has been greatly developed. It also promotes the prosperity of China's beverage industry.
 
Judging from the sterilization methods of packaging materials, there are two main types of ultra-clean hot filling production lines: one is the wet ultra-clean hot filling production line, which mainly uses peracetic acid to sterilize bottles and caps; The other is dry sterilization of bottle embryo (using vaporized hydrogen peroxide to sterilize bottle embryo, hereinafter referred to as dry sterilization of bottle embryo), which is directly filled after bottle blowing, and the cap is sterilized by hydrogen peroxide steam or peracetic acid. Relatively speaking, the wet ultra-clean hot filling production line is safer than the bottle blank dry blowing and filling integrated ultra-clean hot filling production line, but the production energy consumption is relatively higher.
 
Regarding the different filling methods of beverages, domestic experts and scholars mainly compared and discussed the hot filling and aseptic cold filling technologies, and made a preliminary study on the technological requirements of ultra-clean filling, but there was no report on the microbial evaluation of the whole line of the ultra-clean hot filling production line of bottle embryo dry sterilization (blowing, filling, sealing, hereinafter referred to as blowing and filling machine). The author has nearly ten years of experience in the application and management of the dry sterilization of bottle embryos and has a deep understanding of the possible microbial risks in this production line. In order to make a comprehensive microbial evaluation of the bottle embryo dry sterilization blow-filling-sealing integrated ultra-clean hot filling production line, so as to clarify the main microbial risks of this kind of production line, this paper conducts the microbial evaluation of equipment surface smearing, space microbial detection, bottle embryo sterilization challenge experiment, irrigation experiment, etc. from the bottle embryo production to the end of filling and sealing, so as to clarify the possible microbial risks of this production line, remind users to strengthen the management of microbial risks, and provide a reference for peers.
 
Materials and Methods
 
1. Materials and Reagents

Sterilized saline cotton swab Zhengzhou Qizhi Bio; 37℃ microbial incubator and 28℃ microbial incubator Memmert ICP600, Germany.
 
2. Instruments and Equipment

MAS-1OO ECO MERCK, a planktonic sampler; Dust detector 9306-02MK MERCK Company; Bottle embryo injection molding machine NETSTAL Company; Bottle embryo conveyor belt, bottle embryo heating furnace, bottle blowing machine, ultra-clean hot filling machine Krones Company.
 
3. Methods

(1) Microbiological smearing and detection: After soaking with a sterile cotton swab in sterilized physiological saline, smear the target position, and then divide the physiological saline containing cotton swab into two parts, one for the total number of colonies and the other for the detection of mold and yeast.
 
(2) Microbial culture: m-TGE culture medium is used for the total number of colonies, and it is cultured in a 36℃ incubator for 2 days; Yeast was cultured in the m-Green medium at 28℃ for 5 days.
 
(3) Dust particle detection: use the dust particle detector to detect dust particles at each point in the hundred-level space of the filling machine, and the air sampling volume is 28.3L.
 
(4) Detection of settling bacteria and planktonic bacteria: detection of settling bacteria, 30min; at each point; Detection of planktonic bacteria, the air sampling volume at each point is 1m3.
 
(5) Microbiological detection of bottle embryos and bottles: After sampling bottles (embryos), take 10 or 20 bottles as a group, respectively, moisten and wash the inside and outside of the bottles (embryos) with sterilized production saline, then carry out suction filtration, and then respectively carry out colony count and mold yeast culture.
 
(6) Detection of sterilization efficiency of bottle embryo: inoculate the bottle embryo with Bacillus subtilis, sterilize the bottle embryo with hydrogen peroxide dry method according to the normal production process, then punch out the bottle embryo, put it into a sterile bag, wash the bottle embryo with sterile water in clean bench, and carry out suction filtration, detect the number of residual Bacillus subtilis in the bottle embryo, and calculate the sterilization efficiency.
 
(7) Irrigation experiment: According to the normal production process, the bottle embryo is sterilized, blown into the bottle, filled with water in a filling machine, and then sealed with a sterilized cover to obtain an irrigation sample. Samples were filtered on a clean bench to detect microorganisms.
 
Results and Analysis

1. Microbial risk in the production process of bottle embryo

Generally, the production of bottle embryo includes injection molding, cooling, embryo taking, transfer and transportation, which mainly involves injection head, injection mold, embryo taking mold and conveyor belt. In order to clarify the possible microbial contamination in the process of bottle embryo production, the equipment involved in each process of bottle embryo production was smeared in this study, and the results are shown in Table 1.
 
Table 1 Microbiological evaluation of the preform system  
From the above results, it can be seen that microbial colonies can be detected on the surface of equipment in all aspects of bottle embryo production, accounting for about 10% ~ 20%. At the same time, the pollution of mold and yeast on the equipment surface is serious, and the detection rate is as high as 10% ~ 60%. Therefore, in order to control the initial microbial load of bottle embryos, it is necessary to strengthen the cleaning and disinfection of the surface of bottle embryo production equipment and reduce the microbial load of bottle embryos from the source.
 
2. Microbial risk in the process of bottle embryo transportation

In order to find out the initial microbial load of bottle embryos and the pollution during transportation, the microbial contents inside and outside the newly produced bottle embryos and those inside and outside the bottle embryos after transportation in each link were detected, and the results are shown in Table 2.
 
Table 2 Microbiological evaluation of preforms conveying  
From the above test results, it can be seen that there is basically no microorganism detected in the newly produced bottle embryo, but there are microorganisms detected outside the bottle embryo. After the bottle embryo is transported by the conveyor belt, the pollution outside the bottle embryo is more serious, and the detection rate of microorganisms outside the bottle embryo gradually increases, even reaching 100% detection. In addition, from the experimental results, the internal and external pollution of stock bottle embryos is serious. Therefore, in the production process, the initial microbial load of the bottle embryo should be controlled.
 
3. Microbial risk of bottle embryo conveyor belt

From the above experiments, we already know that the microbial contamination of the bottle embryo after transportation is serious, so it is necessary to conduct a comprehensive microbial detection and evaluation of the bottle embryo conveyor belt. The evaluation results are shown in Table 3.
 
Table 3 Microbiological evaluation of preforms conveyor belt  
From the test results, the pollution of bottle embryo conveyor belts is serious, especially the pollution of mold and yeast, which has a great impact on the quality of products. Therefore, the microbial management of bottle embryo conveyors should be strengthened.
 
4. Microbial risk inside bottle blowing machine

Because there are no cleaning and disinfection measures for bottles after blowing, the microbial hygiene inside the bottle blowing machine will directly affect the quality and safety of the filled products. Therefore, the evaluation of the microbial conditions inside the bottle blowing machine is helpful to the quality control of the products produced by the bottle blowing machine. See Table 4 for the microbial evaluation results inside the bottle blowing machine.
 
Table 4 Microbiological evaluation of the blow molding machine  
From the test results, microorganisms were detected on the mold surface and nozzle of the bottle blowing machine. Therefore, for the ultra-clean hot filling production line of bottle embryo dry sterilization, even if the bottle embryo sterilization can meet the requirements, in the process of bottle blowing, if there are no good microbial control measures, it will still cause secondary pollution to the empty bottle.
 
5. Microbial risk inside the filling machine

The blowing-filling-sealing integrated ultra-clean filling machine is equipped with a reliable COP/SOP system to clean and disinfect the inside, and at the same time, a high-efficiency filter is used to filter the air entering the filling machine, so that the internal environment can reach a hundred-level space standard, and the aseptic filling environment can be better achieved. Table 5 shows the evaluation results of the internal microbial smear of Grade 100.
 
Table 5 Microbiological evaluation of isolator of the filler  
From the test results, the surface of the hundred-level space equipment of the filling machine is not sterile. As a matter of fact, microorganisms can be detected on the surface of hundreds of internal equipment of filling machines even when comprehensive COP/SOP is carried out. Therefore, in the production process, it is necessary to pay attention to the parameters such as the concentration of disinfectant in SOP and the action time and adjust the angle and pressure of the COP/SOP nozzle according to the detection results to reduce the probability of microbial contamination.
 
6. Hygienic microbial risk in bottle blowing machine and filling machine space

At present, the bottle blowers in the dry blowing, filling and sealing integrated ultra-clean hot filling production line of bottle embryo are not sterile bottle blowers, and the space of bottle blowers is not completely closed. Only the filtered air blown from the top is used to keep the environment relatively clean. In addition, in the production process, it is often necessary to open the safety door for maintenance due to other reasons such as bottle blowing failure maintenance. Therefore, the sanitary microorganism pollution in the space of bottle blowers is serious. The test results are shown in Table 6. The hundred-level space of the filling machine is protected by a positive pressure control system, COP/SOP and other measures. Relatively speaking, the hygiene of the hundred-level space of the filling machine can basically meet the production requirements of ultra-clean hot filling. The test results are shown in Table 7. From the detection of dust particles, the dust particles in a hundred-level space meet the standard.
 
Table 6 Microbiological evaluation of the space of blow molding machine  
Table 7 Microbiological evaluation of the isolator of the filler  
7. Study on sterilization efficiency of bottle embryo

The sterilization efficiency of the bottle embryos is the key to ensuring the normal production of the ultra-clean filling machine, so the sterilization effect of hydrogen peroxide on bottle embryos directly determines the reliability of the equipment. The experimental results show that the sterilization efficiency of dry hydrogen peroxide on bottle embryo of the blowing-filling-sealing integrated dry sterilization line for ultra-clean hot filling is more than 4 logs, which can basically meet the production process requirements of ultra-clean hot filling. However, the further experimental results show that for normal bottle embryos, after sterilization with hydrogen peroxide, the microbes in bottle embryos were detected immediately, and about 1.6% of bottle embryos still had microbes detected. Therefore, it is very important to control the hygiene of all links of the whole line.
 
8. Experimental study on irrigation

After the normal 4P(COP/CIP/SIP/SOP) cleaning and disinfection of the filling machine, the bottle embryo is sterilized, then the bottle is blown, filled with water (sterile water after cooling), and the cap is sealed after disinfection. Then, the filled sample is filtered to detect microorganisms, and the detection rate of microorganisms is about 3% ~ 5%, which basically meets the technological requirements of ultra-clean hot filling production.
 
Conclusion
 
The pollution of bottle embryos on the conveyor belt is serious, and the pollution is mainly caused by mold. Hygiene and cleanliness of the internal space of bottle-blowing machines and the surface of equipment have a great influence on the pollution in the process of bottle-blowing and empty-bottle transmission. Attention should be paid to hygiene management and daily cleaning and disinfection of the space and surface of the bottle blowing machine. The hundred-level space of a filling machine can't guarantee sterility in a production cycle, so it's very important to make SOP of space regularly. The sterilization of bottle embryos with hydrogen peroxide can only reach the sterilization level of 4log, and the detection rate of microorganisms in sterilized bottle embryos is about 1.6%. Therefore, when producing sensitive products, special attention should be paid to the filling temperature and filling environment to prevent the product quality problems caused by the superposition of various pollution factors.