Method for preserving liquid food using pulsed electric field treatment

Abstract

The present application provides a method for preserving liquid food by means of pulsed electric field treatment. The invention relates to a method for rapidly and uniformly heating a liquid product to a predetermined temperature by means of resistive heating. According to the invention, based on the application of an electric field strength between 0.1 - 5.0 kV / cm for an extended period of time, sufficient and effective microbial inactivation is achieved by selecting a relatively low electric field strength and a pulse duration of at least 10 microseconds, while the maximum temperature of the liquid product during resistive heating is autonomously kept below 92 °C. The method of the invention is effective at neutral pH and at a pH of less than 7. Furthermore, the method of the invention is effective in inactivating a broad range of relevant microorganisms. The invention also relates to the method, wherein the liquid product is pre-heated prior to subjecting the method to the liquid product. The invention also relates to a liquid product obtainable by the method according to the invention.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680066218.4, entitled "Method for Preserving Liquid Food by Using a Pulsed Electric Field", filed on November 17, 2016. Technical Field

[0002] This invention relates to a method for rapidly and uniformly heating a liquid product to a predetermined temperature using resistance heating. The invention also relates to a method wherein the liquid product is preheated before the method is applied to it. Background Technology

[0003] Pulsed electric field (PEF) is a technique used to induce electroporation of cell membranes by applying short-duration pulses of a high-intensity external electric field. The most widely accepted theory for this phenomenon is that applying an external electric field to the biological membrane induces local instability in the phospholipid bilayer, ultimately leading to pore formation. Pore formation (electroporation) promotes permeability across the membrane (electroperfusion), which, depending on the strength of the applied electric field, can be a reversible process or, when applied at high voltages, irreversible, leading to cell death. In continuous-flow PEF treatment systems, different critical times (Mastwijk) must be considered. et al. The total (effective) treatment time is defined as the product of the number of pulses and the time of each pulse received by the fluid element under high electric field conditions as it is pumped through the treatment device. Currently, total treatment time and electric field strength are considered key factors determining the efficiency of irreversible electroporation (Saulis and Wouters, 2007). When using pulses with a duration of 2 microseconds and a total treatment time of 100-400 microseconds, irreversible electroporation is effective for nutrient microorganisms at electric field strengths in the range of 10-20 kV / cm. Figure 1A and Figure 1B Reynard and colleagues (1998) investigated the critical effect of pulse duration for single pulses used in gene transfer. They found that the minimum pulse time required for orientation was ~1 ms and indicated that the critical response time for permeation using a 24 ms pulse at an electric field strength of 1–2.7 kV / cm was 3 to 5 ms.

[0004] In contrast to direct current (DC) pulses, alternating current (AC) is used to induce high electric field conditions in liquids. While an AC current with a fixed frequency (f) can be considered a pulse with a duration of 1 / f, the characteristic pulse waveform considered here is rectangular, meaning the pulse repetition frequency is less than the bandwidth (1 / pulse duration). US 2010 / 0297313 considers AC currents at frequencies greater than 1 MHz (or pulse durations less than 1 microsecond).

[0005] The choice of specific processing conditions depends on the purpose and different applications of electroporation. Reversible electroporation is a step frequently used in molecular biology and clinical biotechnology to introduce small or large molecules into cells, i.e., drugs, oligonucleotides, antibodies, and plasmids, into the cytoplasm with the aim of maintaining cell viability. Irreversible electroporation can be used to extract molecules from cells or to inactivate cells. In this invention, we aim to use irreversible electroporation as a non-thermal preservation method, where the highest temperature and holding time obtained by PEF processing are less than those of conventional heat pasteurization. This results in better preservation of the product's fresh taste and nutritional value, among other benefits. When the purpose is microbial inactivation, the selection of processing conditions for pulsed electroporation depends on several factors, but can be categorized into three groups: process parameters, microbial characteristics, and processing matrix characteristics.

[0006] In addition to electric field strength and treatment time, temperature is also considered crucial for the effectiveness of microbial inactivation via PEF (Raso). et al. (2014). Increasing the electric field strength and processing time will increase the lethality of PEF. Due to these conditions, more energy will be applied per unit mass, resulting in a higher product temperature rise. Typical processing conditions for irreversible electroporation are in the range of microsecond short pulses at high voltages (5-80 kV / cm). Even within the temperature range where microorganisms are not lethal, increasing the temperature of the matrix (e.g., liquid food products) before PEF treatment promotes the degree of microbial inactivation by PEF. Not wishing to be bound by theory, this preheating effect has an impact on the phospholipid bilayer structure of cell membranes, making cells more susceptible to PEF processing (Wouters). et al. , 1999).

[0007] Microbial characteristics influence the effectiveness of PEF in inactivation of microorganisms. Generally, relatively large microorganisms have been reported to be more sensitive to PEF than smaller microorganisms, and Gram-negative microorganisms are more sensitive to PEF than Gram-positive microorganisms.

[0008] The efficacy of PEF treatment is often studied in liquid matrices containing suspended microorganisms. The properties of the treatment matrix have been investigated, and pH has been reported to be crucial to the effectiveness of the treatment. That is, PEF is more effective in low-pH matrices than in neutral-pH matrices.

[0009] Commercial applications of irreversible PEF processing aim to inactivate microorganisms in a continuous stream through a single-pass treatment unit. The practice of providing a loop of more than one pass through the treatment unit by mixing treated and untreated products, as described in US 2012 / 0103831, is circumvented due to the complexity of the processing.

[0010] As mentioned earlier, external pulses are applied to the product, introducing energy and causing a temperature rise. This temperature rise depends on the chosen processing conditions and product characteristics (Heinz et al., 2002). To avoid overheating the product, a cooling section is placed between two processing chambers in some applications (Sharma et al., 2014); however, this method requires more electrical energy and energy for cooling. Another possibility described for avoiding overheating is to introduce a pause after the application of the pulse or after a series of pulses (El Zakhem). et al. (El Zakhem et al., 2006); however, this is not feasible in commercial applications because the total processing time in this study increased to 5200 s–7800 s (El Zakhem et al., 2007), while the typical time for online heat pasteurization is in the range of seconds to minutes. A pause of at least one minute between pulses or between a series of pulses is also applied in the batch system described in CA 2758678. Commercially applied processing conditions utilize PEF with an electric field strength between 10 and 30 kV / cm, as applying higher electric field strengths has technical limitations and may cause dielectric breakdown of the food.

[0011] The applied processing conditions are suitable for liquid food products with low pH, i.e., highly acidic fruit juices with a pH below approximately 4.6. These processing conditions are present in several applications where there is a need to adapt them for inactivating larger-sized microorganisms in liquid food products. Furthermore, Gram-negative microorganisms can be inactivated more effectively than Gram-positive microorganisms. In particular, inactivating small-sized Gram-positive bacteria is problematic in most cases under currently known PEF processing conditions. Moreover, current low-pH process conditions are not an efficient approach suitable for food products with a pH above approximately 4.6. Current PEF processing for liquid food products involves relatively high electric field strengths, i.e., 5 kV / cm and higher, typically 10–30 kV / cm. These relatively high electric field strengths typically hinder PEF processing from being scaled up to large volumes due to peak power requirements and limitations on single-pulse duration through maximum stored pulse energy. These technological boundaries limit the maximum production capacity of a single line used for preserving low-conductivity acidic fruit juices to 5000 L / h.

[0012] Thus, there is a need for PEF processing conditions as follows: - Effectively inactivates Gram-positive bacteria and / or microorganisms of relatively small size, preferably without loss of efficiency in inactivating Gram-negative bacteria and / or microorganisms of relatively large size; and / or - Inactivate microorganisms and / or spores in liquid food products with a pH above approximately 4.6 and a pH below 4.6; and / or - More commercially viable than existing inactivation technologies; and / or - Applicable and has the potential to scale up to a production capacity greater than the existing state of the conditions in the field used using a single line. Summary of the Invention

[0013] This invention relates to a method for rapidly and uniformly heating a liquid product to a predetermined temperature using resistance heating to obtain a heated liquid product, comprising: (a) Providing liquid products; (b) Provide an apparatus for rapidly and uniformly heating a liquid product to a predetermined temperature using resistance heating; (c) Continuously supplying the liquid product to the inlet of the device and allowing the liquid product to flow through the device; (d) Continuously generating an electric current through the liquid product flowing in the device, wherein at least one pulse is applied to each fluid element during the passage, the pulse duration is at least 10 microseconds, and the electric field strength is 0.1 to 5 kV / cm; and During the resistance heating, the highest temperature of the liquid product autonomously remains below 92°C.

[0014] Part of this invention lies in the fact that the pulse duration of a single pulse is a critical factor, rather than the total effective processing time. At a pulse duration of 2 microseconds (τ) and an electric field strength (E) of 10 kV / cm, the inventors found that inactivation was not effective, even though the total effective processing time was calculated to be E. 2 τ is four times that at 20 kV / cm using conventional PEF treatment. Under conditions of 0.1–5 kV / cm, inactivation was found to be effective only for pulse durations exceeding 10 microseconds, for example, between 100 and 1000 microseconds.

[0015] The method of this invention is applicable to liquid food products and liquid feed products, and the PEF processing conditions of this invention are equally effective in inactivating both Gram-negative and Gram-positive bacteria. The PEF processing conditions are applicable to liquid food products and liquid feed products, wherein these conditions are effective in inactivating both relatively large and relatively small microorganisms. Furthermore, the inventors have surprisingly discovered PEF processing conditions now applicable to current application conditions at relatively low pH levels, as well as those applicable to higher pH conditions. Finally, the inventors have discovered that processing conditions suitable for higher production volumes of liquid food or liquid feed products can be applied compared to prior art methods of rapidly and uniformly heating liquid products to a predetermined temperature via resistance heating.

[0016] A second aspect of the invention relates to liquid products that can be obtained by the method according to the invention. Attached Figure Description

[0017] Figure 1A , Figure 1B After various PEF treatment conditions, E. coli (E. coli) were found in orange juice at pH 3.8. Escherichia coli Listeria monocytogenes ( Listeria monocytogenes Lactobacillus plantarum ( Lactobacillus plantarum Salmonella Sanfutonburg ( Salmonella Senftenberg ), brewer's yeast ( Saccharomyces cerevisiae The reduction in viable cell count. The left image represents the currently used PEF conditions, while the right image shows the PEF conditions of the present invention. Figure 1BBelow each image is a reference for various PEF processing conditions related to that image. Solid black triangle: 10 kV / cm, 2 microseconds; Solid gray rhombus: 15 kV / cm, 2 microseconds; Hollow white circle: 20 kV / cm, 2 microseconds; Solid gray circle: 0.9 kV / cm, 1000 microseconds; Solid black rhombus: 2.7 kV / cm, 1000 microseconds; Hollow white rhombus: 2.7 kV / cm, 100 microseconds; Dashed line: Detection limit.

[0018] Figure 2 Temperature-conductivity distribution of orange juice (pH 3.8), coconut water (pH 5.0), and watermelon juice (pH 6.0).

[0019] Figure 3A , Figure 3B After PEF treatment at 2.7 kV / cm and 1000 microseconds, E. coli in orange juice, coconut water, and watermelon juice ( E. coli ) and Listeria monocytogenes ( L. monocytogenes The decrease in viable bacterial count.

[0020] Figure 4A , Figure 4B Microbiological analysis of untreated and PEF-treated orange juice (n=6), some of which were qualitative. Figure 4B The rest are quantitative. Figure 4A ).

[0021] Figure 5A , Figure 5B Sensory evaluation of orange juice samples stored at 7°C and ambient temperature within the indicated time period, where samples were rated “good” if they were comparable to freshly squeezed orange juice, and “bad” if they were not comparable to freshly squeezed orange juice.

[0022] Figure 6 : The amount of soluble solids in orange juice stored at 7°C and ambient temperature for 3 months before and after PEF treatment (°Brix).

[0023] Figure 7 Acidity of orange juice before and after PEF treatment during storage at 7°C and ambient temperature for 3 months.

[0024] Figure 8 pH of orange juice before and after PEF treatment during storage at 7°C and ambient temperature for 3 months.

[0025] Figure 9 Oil content of orange juice stored at 7°C and ambient temperature for 3 months before and after PEF treatment.

[0026] Figure 10 Vitamin C content of orange juice stored at 7°C and ambient temperature for 3 months before and after PEF treatment.

[0027] Figure 11 Pectin esterase activity during storage at 7°C and ambient temperature for 3 months before and after PEF treatment. Detailed Implementation

[0028] The inventors have now discovered that PEF processing conditions are suitable for liquid food products and liquid feed products, wherein these conditions are equally effective in inactivating both Gram-negative and Gram-positive bacteria. The inventors have also discovered that PEF processing conditions are suitable for liquid food products and liquid feed products, wherein these conditions are effective in inactivating both relatively large and relatively small microorganisms. Furthermore, the inventors have surprisingly discovered that PEF processing conditions applicable to both relatively low and higher pH conditions are now applicable. Finally, the inventors have discovered that PEF processing conditions can be applied to achieve higher production volumes for liquid food and liquid feed products compared to the quantities achievable with currently available processing methods.

[0029] Therefore, the inventors have provided a method to overcome many disadvantages associated with currently known methods for heating liquid products to obtain liquid products with reduced microbial load.

[0030] This invention relates to a method for rapidly and uniformly heating a liquid product to a predetermined temperature using resistance heating to obtain a heated liquid product, comprising: (a) Providing liquid products; (b) Provide an apparatus for rapidly and uniformly heating a liquid product to a predetermined temperature using resistance heating; (c) Continuously supplying the liquid product to the inlet of the device and allowing the liquid product to flow through the device; (d) Continuously generating an electric current through the liquid product flowing in the device, wherein at least one pulse is applied to each fluid element during passage, the pulse duration is at least 10 microseconds, and the electric field strength is 0.1 to 5 kV / cm; and During the resistance heating, the highest temperature of the liquid product autonomously remains below 92°C.

[0031] According to the present invention, a method for rapidly and uniformly heating a liquid product to a predetermined temperature by means of resistance heating provides a heated liquid product with reduced microbial load.

[0032] Heating liquid products to temperatures above a predetermined maximum temperature can lead to undesirable reductions in fresh flavor, vitamins, and nutrients, as well as denaturation of proteins present in fresh (untreated) products. The degree of component reduction and denaturation is related to the temperature and time the product is subjected to. Different liquid products are subjected to different temperature-time combinations to achieve the desired degree of enzyme and microbial inactivation. Alternative (non-)thermal methods of reducing product temperature and / or exposure time are therefore of great interest because they can better preserve the freshness characteristics of the product. When the temperature can be reduced or the exposure time can be shortened, better product quality can be expected. In the method of the present invention, the heating exposure time is greatly reduced, and due to the selected processing conditions, the maximum temperature of the liquid product is autonomously maintained below about 92°C during resistance heating. Preferably, according to the method of the present invention, the maximum temperature of the liquid product is autonomously maintained below a critical temperature during resistance heating at which the liquid product does not suffer from reduction of heat-sensitive components (if present in the product) or denaturation of proteins (if present in the product), while the microbial load in the liquid product is reduced to an acceptable target level. Now, thanks to the method of the present invention, processing conditions have become applicable, which both prevents the liquid product from overheating and effectively and efficiently reduces the microbial load of the liquid product.

[0033] In the method according to the invention, the pulse duration of a single pulse is a critical factor, not the total effective treatment time. When a pulse duration of 2 microseconds and an electric field strength of 10 kV / cm are applied, microbial inactivation is determined to be ineffective, even though the total effective treatment time is four times that of conventional PEF treatment at an electric field strength of 20 kV / cm. It is undesirable to be bound by theory and interpret this as the electroporation effect being disrupted at the reduced electric field strength of 10 kV / cm.

[0034] The inventors have now surprisingly discovered that microbial inactivation is effective under conditions of low electric field strengths of 0.1-5 kV / cm, combined with extended pulse durations of 100 and 1000 microseconds, achieving inactivation at a lower intensity than the temperature-time combination required for conventional heat pasteurization or PEF treatment at 10 kV / cm. Not wishing to be bound by theory, these findings indicate that pulse duration has become a key factor in the method according to the invention.

[0035] To avoid being bound by theory, the external electric field applied to the product affects protein channels in the cell membrane and / or the lipid domains of the cell membranes of microorganisms present in the product, leading to conformational changes in the channels and / or domains. Membrane protein channels open at a membrane potential of 50 mV, which is far below the 150-400 mV required for pore formation in the lipid bilayer (Tsong, 1992).

[0036] Since the opening and closing of many protein channels depends on transmembrane potential, it is assumed that voltage-sensitive protein channels will be opened when electrical treatment is applied. Once these channels are opened, they conduct currents larger than those designed for them. As a result, these channels can be irreversibly denatured by Joule heating and / or electrical modification of their functional groups (Tsong, 1992). The opening / closing of protein channels occurs in the submicrosecond timescale, while protein denaturation takes milliseconds to seconds (Tsong, 1992).

[0037] This indicates that protein channels can be affected by electric field strengths 3 to 8 times less than those affecting the lipid bilayer; that is, electric field strengths between 2.5 kV / cm and 7 kV / cm are used to inactivate protein channels compared to the 20 kV / cm required for irreversible damage through lipid bilayer electroporation (i.e., conventional PEF conditions). According to the invention, an electric field strength between 0.1 kV / cm and 5 kV / cm combined with a pulse duration of 10-1000 microseconds is sufficient and effective for establishing effective microbial inactivation in liquid products. For example, the method of the invention is applicable to liquid products in a 1 L / h PEF device (“PEF system”). For the illustrated liquid product, the pulse duration is set to 100 microseconds or 1000 microseconds, and the selected electric field strength is 0.9 kV / cm or 2.7 kV / cm. The number of pulses applied to the liquid product varied between 0 and 35, the time interval between two consecutive pulses varied between 0.6 milliseconds and 199 milliseconds, and the highest temperature obtained varied between 36°C and 92°C. See the following example demonstrating the efficiency of the method of Embodiment 1 of the present invention for a more detailed overview.

[0038] The inventors have now discovered that microbial inactivation is particularly effective for vegetative cells. Most likely, based on this effect of the method of the present invention, the method according to the invention also provides an effective mechanism for inactivating spores. Spores contain essential proteins for germination in the inner membrane and sporodermis, and these proteins are targeted by external electrical stimulation.

[0039] As another example of applying the method of the present invention, for instance, in processing a batch of liquid products using the method of the present invention, a 1200 L / h PEF device is used to rapidly and uniformly heat the liquid products to a predetermined temperature by means of resistance heating. The pulse duration is 1000 microseconds and the electric field strength is 2.0 kV / cm. The number of pulses applied to the liquid products is approximately 5 pulses, and the time interval between two consecutive pulses is 3.8 milliseconds. See also Example 3 below for a more detailed embodiment of the present invention.

[0040] One embodiment of the invention is a method according to the invention, wherein the pH of the liquid product is between pH 1.5 and 9.0, preferably above 4.6, preferably between 4.8 and 9.0, more preferably between 5.5 and 8.0, and even more preferably between 6.0 and 7.5. Another embodiment of the invention is a method according to the invention, wherein the pH is above about 5.0, preferably about 6.0.

[0041] In another embodiment, the present invention relates to a method according to the invention, wherein the pH of the liquid product is below 4.6, preferably between 1.5 and 4.6, more preferably between about 1.5 and about 3.8.

[0042] In another embodiment of the invention, in the method according to the invention, the pH of the liquid product is higher than 4.6, preferably between 4.6 and 9.0. One embodiment of the invention is a method according to the invention wherein the pH of the liquid product is between 5.0 and 9.0, preferably between 6.0 and 9.0.

[0043] Now, due to the applicability of the method of the present invention, liquid products with a wide range of pH can be processed in one and the same methods according to the invention. Because the method of the present invention is applicable to processing liquid products with such a wide range of pH, the diversity of liquid products desired for and selected for processing in the method of the present invention is very large. In fact, any liquid product, raw material, or semi-finished product applied, for example, in food processing, is now suitable for rapid and uniform heating by the method of the present invention. Since the inventors have surprisingly found their PEF method to be effective and efficient over such a wide pH range, the processing of food products using methods incorporating PEF according to the invention has now become more widely applicable and easier to use than before.

[0044] Furthermore, one embodiment of the invention is a liquid product according to the invention, wherein the liquid product has a conductivity between 0.01 and 10 S / m measured at 20°C, more preferably between 0.1 and 3 S / m measured at 20°C, and most preferably between 0.2 S / m and 0.8 S / m measured at 20°C.

[0045] Typically, conductivity within these indicated boundaries is preferred because such conductivity facilitates rapid and uniform heating of liquid products according to the method of the invention. Since most liquid food products have conductivity within boundaries suitable for applying the method of the invention to said liquid products, the method of the invention is suitable for processing a wide variety of liquid food products for which reduced microbial load is desired. For example, the conductivity of several batches of liquid food products at 20°C has been measured, and for example, cranberry juice is 0.1 S / m, beer is 0.15 S / m, apple juice is 0.2 S / m, chocolate milk is 0.4 S / m, whole milk is 0.45 S / m, soy milk is 0.4 S / m, almond milk is 0.25 S / m, carrot juice is 1.0 S / m, and tomato sauce is 1.8 S / m. Thus, the method of the invention is applicable to a wide variety of liquid food products.

[0046] Not wanting to be bound by theory, the PEF processing conditions of this invention seem to open up a path to new mechanisms for microbial inactivation.

[0047] The inventors have now discovered that when the PEF processing conditions of the present invention are applied, including an electric field strength of surprisingly low 0.1-5 kV / cm, preferably 4 kV / cm or lower, more preferably 3 kV / cm or lower, combined with a pulse duration of 10-1000 microseconds, preferably about 1000 microseconds, more preferably about 100 microseconds, and at a maximum temperature of the liquid food product between 40°C and 92°C, preferably between 50°C and 92°C, more preferably between about 60°C and 85°C, the liquid food product is effectively pasteurized, i.e., the microorganisms are effectively and efficiently inactivated.

[0048] According to the present invention, pasteurization based on the method of the present invention is particularly efficient and effective when the number of pulses applied to each fluid element during passage through the processing zone is at least 1, preferably 1 to 100, more preferably 5 to 50.

[0049] The number of pulses is given by Equation 1, where n It is the number of pulses. V It is the volume of the processing chamber (L). f The pulse frequency (Hz) used, and Flow rate (L / h): (Equation 1) According to the present invention, the number of pulses is not a critical step in designing the method, provided that at least one pulse is applied to each fluid element flowing through the processing chamber of the apparatus for rapidly and uniformly heating a liquid product. To ensure that at least one pulse is applied to each fluid element, the system must be designed with the aim of ensuring a residence time in the processing chamber greater than 1 / f. The number of pulses applied will be a result of the method design, based on the desired production volume of the liquid product obtained using the method. The following parameters were considered: liquid product conductivity (σ, S / m), specific heat capacity (cp, kJ / kg-K), density (ρ, kg / m3), applied electric field strength (E, V / m), and pulse duration (τ). puise The parameters are , s) and the temperature gradient (ΔT, °C) (the difference between the inlet and outlet temperatures of the liquid product). The relationship between these parameters is given by Equation 2.

[0050] (Equation 2)

[0051] As previously mentioned, and further illustrated in Example 1 below, the pulse duration is crucial to the effectiveness of PEF processing according to the method of the present invention. Thus, the application of a relatively long pulse can be more effective than the application of a shorter pulse with a similar total effective processing time.

[0052] For example, for a liquid product processed at 1 L / hr in a continuous flow 1 L / h PEF device according to the method of the present invention, typically, the pulse duration is about 100-1000 microseconds, and the electric field strength is about 2.7 kV / cm. Typically, the number of pulses is then about 1-25, and the time interval between two consecutive pulses is about 0.6-39 milliseconds, depending on the desired temperature increase through the processing chamber, i.e., the difference between the inlet temperature and the maximum temperature.

[0053] For example, in the method according to the invention, for a batch of liquid product processed in a 1200 L / h PEF device according to the method of the invention, typically, the pulse duration is 1000 microseconds and the electric field strength is about 2.0 kV / cm. Typically, the number of pulses is then about 5, and the time interval between two consecutive pulses is about 3.8 milliseconds.

[0054] Typically, according to the present invention, the method of the present invention is applicable to processing liquid products in PEF equipment having a production capacity between 30 L / h and 200 L / h.

[0055] Typically, according to the present invention, the method of the present invention is applicable to processing liquid products in a PEF equipment having a production capacity of about 30,000 L / h.

[0056] One embodiment of the invention is the method of the invention, wherein the equipment for rapidly and uniformly heating a liquid product to a predetermined temperature has a production rate between about 1 L / h and about 30,000 L / h, preferably about 1 L / h or about 30 L / h, or about 200 L / h, or about 1200 L / h, or about 30,000 L / h.

[0057] Furthermore, not wanting to be bound by theory, the PEF processing conditions of this invention follow the theoretical mechanism of inactivating protein channels in cell membranes, leading to microbial inactivation. Compared to currently used processing conditions for PEF, these novel PEF processing conditions of this invention offer new opportunities for microbial inactivation. Using these new conditions, all nutrient microorganisms are inactivated, without preference for relatively large microorganisms and / or for Gram-negative microorganisms compared to relatively smaller microorganisms and / or Gram-positive microorganisms.

[0058] Thus, using the method of the present invention, Gram-negative microorganisms such as, for example, Escherichia coli can be inactivated in liquid products. Escherichia coli ) strains, Salmonella spp. Salmonella ) bacterial strains, other Enterobacteriaceae ( Enterobacteriacea ) and acetic acid bacteria. Additionally, the method of the present invention also inactivates Gram-positive microorganisms such as, for example, Listeria monocytogenes in liquid products. Listeria monocytogenes Lactobacillus plantarum ( Lactobacillus plantarum Leuconostoc ( ) Leuconostoc ) strains and Streptococcus spp. Streptococcus ) bacterial species. Based on the expected theoretical mechanism, it is also expected that spore-forming bacteria can be inactivated, because their cell membranes also contain voltage-gated channels, such as *Bacillus cycloalis* spp. ( Alicyclobacillus Bacteria and Clostridium ( Clostridium Bacteria. In addition, the spore itself contains essential proteins for germination in the inner membrane and sporodermis, which can be targeted by externally applied pulses.

[0059] Furthermore, the method of the present invention is applicable to the inactivation of relatively large microorganisms in liquid products, such as, for example, yeasts and molds. The method of the present invention is also applicable to the inactivation of relatively small microorganisms in liquid products, such as, for example, Listeria monocytogenes. L. monocytogenes Of course, the method of the present invention is equally applicable to the inactivation of microorganisms in liquid products having sizes between those of these example microorganisms, as shown in Example 1.

[0060] Furthermore, by utilizing these new conditions of the present invention, the inactivation of all types of microorganisms in (liquid food) products is now possible, because the PEF processing conditions of the present invention are equally effective in inactivating microorganisms in liquid food products with relatively low pH and liquid food products with relatively high pH.

[0061] Finally, because the electric field strength used in the PEF processing conditions of this invention is lower than that of currently applied PEF conditions, these conditions of this invention are easily scaled up. This is because the lower peak voltage used in this invention makes the PEF processing conditions of this invention suitable for implementation in industries with large capacity and production volumes. The method of this invention is particularly suitable for liquid food products subjected to the method of this invention in equipment used for rapid and uniform heating of liquid products to a heating temperature by means of resistance heating. The pulse frequency is limited between 1 kHz and 50 kHz to avoid metal release from the electrodes (Mastwijk, 2006). Typically, according to the invention, the flow rate of the liquid product is then between about 1 L / h and 5000 L / h, preferably between about 1000 L / h and 30,000 L / h.

[0062] Another embodiment of the present invention is a method according to any of the foregoing embodiments of the present invention, wherein the liquid product is a liquid food product or a liquid feed product.

[0063] Furthermore, one embodiment of the present invention is a method according to the present invention, wherein the liquid product is a raw material, a semi-finished product, or a final liquid product, such as fruit juice, vegetable juice, baby food, jam, spread or smoothie, alcoholic or non-alcoholic beverage, dairy product, plant-based milk product, liquid egg, soup, or sauce.

[0064] One embodiment of the present invention is a method according to the present invention for rapidly and uniformly heating a liquid product to a predetermined temperature by means of resistance heating, wherein the dairy product is selected from milk, milk products or liquid compositions comprising milk components or milk fractions.

[0065] Furthermore, one embodiment of the present invention is a method according to the present invention, wherein the liquid product is a dairy product comprising milk, milk products, milk components or milk portions.

[0066] According to the invention, an important aspect of the invention is the discovery that during PEF processing, in order to maintain the temperature of the liquid product below about 92°C, or below about 85°C, or below about 70°C, or below about 60°C, no cooling section is required between the processing chambers of the equipment used in the method according to the invention.

[0067] As mentioned above, in the current state of methods in the art, cooling sections are added between processing chambers to prevent the liquid product from overheating. As described, an important finding in this invention is that cooling is not required during the method of the invention because the temperature rise at the critical temperature is very rapid (the residence time in the processing chamber is less than 1 second) and the exposure time at the highest temperature is very short.

[0068] To provide an effective method, as described in this invention, electrical energy is used to expose only to the critical temperature (i.e., the temperature at which product quality will be affected). The non-critical temperature range can be preheated using conventional heating. Therefore, in the method, preferably, the liquid product is preheated to a temperature in the range of 20°C to 70°C, preferably 35°C to 65°C, and more preferably 40°C to 60°C before being supplied to the equipment. One embodiment of the invention is a method according to the invention, wherein the liquid product is preheated to a temperature in the range of 20°C to 70°C, preferably 35°C to 65°C, and more preferably 40°C to 60°C before being supplied to the equipment.

[0069] For practical purposes, liquid products, such as liquid food products, subjected to the method according to the invention are immediately cooled to or below ambient temperature, i.e., cooled to 2-8°C. Since no holding time is required, cooling of the liquid product is performed directly after it leaves the high-field region (preferably within 3 seconds). In the current method design of the invention, a cooling pipe can be installed 0.5 m downstream of the high-field region. For a 30,000 L / h (=8.3 L / s) system with a 3” pipe diameter, this means determining a capacity of 2.2 L or 0.27 seconds before entering the first cooling section and approximately 1-5 seconds (depending on viscosity) before the critical temperature drops by the first 10°C from the highest temperature, followed by cooling to the desired (conventional) outlet temperature, typically in the range of 4-7°C.

[0070] The term “autonomously” has its conventional meaning, and here refers to the temperature of a liquid product that reaches a certain value during processing in the method of the present invention without external cooling (or heating assistance).

[0071] Liquid products, such as liquid food products selected from orange juice, dairy products, coconut water, and watermelon juice, are preheated, for example, to about 40°C, about 50°C, or about 60°C. For example, in order to efficiently and effectively inactivate microorganisms in liquid products based on the method of the present invention, the liquid products are preheated to between about 30°C and 65°C, preferably between 36°C and 59°C.

[0072] According to the invention, preferably, the maximum temperature of the liquid product is autonomously maintained below about 85°C during resistance heating, more preferably below about 70°C, or below about 63°C, or below about 60°C. As part of the invention, the process parameters are selected such that the maximum temperature of the liquid product is autonomously maintained below the selected temperature. When the method of the invention is applied, temperatures equal to or below the selected temperature ensure efficient and effective killing of microorganisms present in the liquid product, while preventing, or at least largely preventing, the loss of undesirable fresh flavor, vitamins, and nutrients, and the denaturation of proteins present in fresh (untreated) liquid products.

[0073] According to the present invention, by applying the electric field strength according to the invention and utilizing the pulse duration according to the invention, the inventors surprisingly discovered that the temperature of the liquid product remains below a maximum temperature of about 92°C, or about 85°C, or about 70°C, or about 60°C, making the method of the invention particularly suitable for implementation in large-scale environments, such as commercial environments. One example of a commercial application of the method of the invention is the processing of liquid food products in an apparatus for rapidly and uniformly heating liquid products to a predetermined temperature using resistance heating, wherein the flow rate of the liquid food product through the apparatus is between about 500 L / h and 30,000 L / h, for example, about 1200 L / h.

[0074] Therefore, one embodiment of the present invention is a method according to the present invention, wherein the temperature of the liquid product is autonomously maintained below 85°C, preferably below 75°C, and more preferably below 60°C during resistance heating.

[0075] In one embodiment of the invention relating to the method according to the invention, the electric field strength is less than about 5 kV / cm.

[0076] One embodiment of the invention is a method according to the invention, wherein the electric field strength is 0.5 to 5 kV / cm, more preferably 2.5 to 4 kV / cm. In one embodiment of the invention relating to the method of the invention, the electric field strength is less than about 3 kV / cm, preferably about 2.7 kV / cm or lower, more preferably between about 0.9 and about 2.5 kV / cm.

[0077] One embodiment of the invention is a method according to the invention, wherein the pulse duration is at least 10 microseconds, more preferably 10 to 2000 microseconds, even more preferably 50 to 500 microseconds, and most preferably 50 to 100 microseconds. In one embodiment of the invention relating to the method according to the invention, the pulse duration is between about 100 microseconds and about 1000 microseconds. In another embodiment of the invention relating to the method according to the invention, the pulse duration is 1000 microseconds or less, preferably about 100 microseconds.

[0078] One embodiment of the invention is a method according to the invention, wherein the applied pulse is a bipolar pulse. Thus, one embodiment of the invention is a method according to the invention, wherein at least one pulse applied to the liquid product is a bipolar pulse. Applying a bipolar pulse to the liquid product is advantageous to avoid electrode damage (Loeffler, 1996). Of course, as part of the invention, other types of pulses are equally applicable to the method of the invention.

[0079] The method of the present invention is particularly applicable to the inactivation of microorganisms in liquid food products such as juices, sauces, and dairy products. That is, examples of such liquid food products are raw materials, semi-finished products, or final liquid products, such as fruit juices, vegetable juices, baby food, jams, spreads or smoothies, alcoholic or non-alcoholic beverages, dairy products, plant-based milk products, liquid eggs, soups, or sauces.

[0080] One embodiment of the present invention is a method according to the invention, wherein the method is for the inactivation of microorganisms in a liquid product. As previously stated, the method of the present invention, using resistance heating for heating liquid products, has many advantages over current methods. One of the main advantages that can be achieved using the method of the present invention is the pasteurization of liquid products, such as liquid food products, wherein the microorganisms are small or large, and wherein the microorganisms are Gram-negative or Gram-positive microorganisms.

[0081] For practical purposes, liquid products, such as liquid food products, subjected to the method according to the invention are immediately cooled to ambient temperature or below, for example, to 2-8°C, after the method of the invention is applied to the liquid product.

[0082] Therefore, one embodiment of the invention is a method in which a heated liquid product is immediately cooled after flowing through a device for rapidly and uniformly heating the liquid product to a predetermined temperature using resistance heating. Of course, for practical purposes, this cooling is applied appropriately and immediately, for example as quickly as possible, preferably within 3 seconds, from the moment the liquid product flows through the device. Thus, another embodiment of the invention is also a method according to the invention, in which a heated liquid product is cooled after being transferred through a device for rapidly and uniformly heating the liquid product to a heating temperature using resistance heating. As part of the invention, the method according to the invention is applicable to applications utilizing a device for rapidly and uniformly heating a liquid product to a heating temperature using resistance heating, the device operating at a low flow rate of about 0.5 L / h to about 2000 L / h, preferably about 0.5 L / h to about 2 L / h, more preferably about 1 L / h, or equivalently preferably about 100 L / h to about 2000 L / h, preferably about 1000 to 1500 L / h, more preferably at about 1200 L / h of liquid product. Preferably, the flow rate is approximately 30,000 L / h.

[0083] As previously stated, prior preheating of the liquid product before subjecting it to a method of rapidly and uniformly heating it to a predetermined temperature using resistance heating improves the inactivation efficiency during processing. However, cooling of the liquid product during processing is a prerequisite because, when applying PEF conditions known in the art, the liquid product is heated to unacceptable values. As mentioned, an important finding now in this invention is that cooling is not required during processing because the temperature of the liquid product does not exceed an unacceptable threshold. Therefore, in the method of this invention, the liquid product is appropriately preheated before subjecting it to a method of rapidly and uniformly heating it to a predetermined temperature using resistance heating, without the need for cooling during processing.

[0084] Thus, in this method, preferably, the liquid product is preheated to a temperature ranging from 20°C to 70°C before being supplied to the equipment, preferably from 35°C to 65°C, and more preferably from 40°C to 60°C.

[0085] The method according to the invention is highly effective and efficient in inactivating microorganisms present in liquid products subjected to treatment by the method according to the invention, which uses resistance heating to rapidly and uniformly heat the liquid product to a predetermined temperature. When the method of the invention is applied to liquid products containing microorganisms, the microbial count is reduced by at least 2 log cfu / mL, most preferably 6 log cfu / mL or more.

[0086] Thus, the present invention is preferably a method in which the microbial count (colony-forming units; CFU) in a liquid product is reduced by at least 2 log CFU / mL, preferably at least 5 log CFU / mL, and most preferably 6 log CFU / mL or more. One embodiment of the invention is a method according to the present invention in which the microbial count in a liquid product is reduced by at least 4 log CFU / mL, preferably at least 7 log CFU / mL.

[0087] The inventors have determined that such a reduction in microbial counts in liquid food products significantly contributes to the preservation of product quality indicators. For example, compared to untreated liquid food products, the taste, aroma, color, and appearance of liquid food products treated by the method of the present invention are preserved for an extended period of time. For instance, when the method of the present invention is applied to orange juice, the taste and aroma of the liquid food product orange juice are preserved for about 60 days or more when stored at about 7°C. Equivalently beneficially, after orange juice is treated by the method of the present invention, the quality of the orange juice is consolidated for about 23 days when maintained at ambient temperature.

[0088] The method of this invention is equally applicable to the inactivation of Gram-positive microorganisms in liquid products, as well as the inactivation of Gram-negative microorganisms in liquid products. Furthermore, the size of the microorganisms is not a limiting factor, meaning that both small and large microorganisms can be inactivated by the method according to the invention. Thus, various aspects and embodiments of the invention represent an important contribution to the art because, until this invention, known methods for rapidly and uniformly heating liquid products to a heating temperature using resistance heating could not efficiently inactivate small Gram-positive microorganisms. Therefore, one embodiment of the invention is the method according to the invention, wherein the microorganisms optionally include Gram-positive microorganisms.

[0089] When the method according to the invention is applied to a liquid product, the pH of the liquid product is changed to a minimum (if any) during the process. This is another advantageous feature of the method according to the invention.

[0090] Therefore, in one embodiment, the present invention relates to a method wherein the pH of a liquid product at the end of the method differs from the pH at the beginning of the method by within 0.5 pH units, preferably within 0.2 pH units, more preferably within 0.1 pH units, and most preferably within 0.05 pH units.

[0091] The method according to the invention is applicable to liquid products, particularly liquid food products.

[0092] A second aspect of the invention relates to liquid products that can be obtained by the method of the invention as described above.

[0093] The present invention is further illustrated by the non-limiting embodiments provided below.

[0094] Example

[0095] Example 1: Escherichia coli (E. coli) in orange juice at pH 3.6 (1 L / h scale) E. coli ), mononuclear cell proliferative plum Sterilella ( Listeria monocytogenes Lactobacillus plantarum ( Lactobacillus plantarum ), Sanfton Castle Sand Pylori ( Salmonella Senftenberg and brewer's yeast ( Saccharomyces cerevisiae inactivation of )

[0096] Pathogenic and spoilage microorganisms were selected based on their morphology, their association with orange juice, and their prevalence in orange juice. Furthermore, heat tolerance and PEF resistance were used as criteria for strain selection. The selected microorganisms are listed in Table 1.

[0097] Table 1. Bacterial and yeast strains used in this example

[0098] Fresh cultures of all five microorganisms listed in Table 1 were prepared using culture media and frozen primary cultures from overnight cultures. *Escherichia coli* (… Escherichia coli Listeria monocytogenes ( Listeria monocytogenes ), brewer's yeast ( Saccharomyces cerevisiae ) and Salmonella Sanftonburg ( Salmonella enterica subsp. enterica serovar Senftenberg's training was conducted by Timmermans et al. As described in 2014. Lactobacillus plantarum (Lactobacillus) was prepared similarly by spreading frozen primary cultures from MRS medium (containing 52.2 g MRS (De Man, Rosoga, and Sharp broth, Merck) and 12 g agar per 1 L of distilled water) onto plates. Lactobacillus plantarum Fresh cultures were inoculated. Petri dishes were incubated overnight at 30°C. A single colony was inoculated into a 100 mL flask containing 100 mL of MRS broth and incubated for 24 h at 20°C in a shaking incubator (180 rpm). 200 μL of this culture was used to inoculate 19.8 mL of fresh MRS broth (100 mL flask) supplemented with 1% glucose and incubated at 20°C and 180 rpm for 24 h.

[0099] After culturing, the cells were washed and a suspension of the selected microorganisms was added to orange juice (Minute Maid). ® (), to a final concentration of approximately 1.0E+8 - 1.0E+9 cfu / mL.

[0100] Prior to electrotreatment, the inoculated suspension was pumped through a 1 L / h PEF system at a flow rate of 13.0 ± 0.5 mL / min and preheated to 36°C. The suspension then entered two vertically positioned collinear treatment chambers where electrotreatment was administered. Due to variations in treatment conditions (electric field strength, pulse duration, and number of applied pulses), the juice autonomously heated to a variable maximum temperature (Table 2 illustrates the conditions used). No holding section was added, so the juice was cooled directly after leaving the treatment chamber (within 3 seconds) by a cooling spiral immersed in a water bath. At the outlet, the sample was aseptically collected. Variations in the selected frequency, the number of applied pulses, and the subsequent temperature rise caused variations in the maximum temperature (Table 2). Samples were collected at different maximum temperatures, and the kinetics of the electric field strength and pulse duration used for fixation were determined.

[0101] The number of viable microbial cells was determined by plate-spreading 100 μL of PEF-treated juice, serially diluted in sterile peptone physiological saline diluent (PSDF), onto suitable agar plates supplemented with 0.1% sodium pyruvate (to promote the growth of sublethal damaged cells). (Timmermans) et al. , 2014). At 25°C (Saccharomyces cerevisiae ... S. cerevisiae ), 30°C (Listeria monocytogenes ()), L. monocytogenes Lactobacillus plantarum ( L. plantarum )) or 37°C (Salmonella Sanfutonburg) S. Senftenberg, Escherichia coli ( E. coli )) Calculate the number of surviving cells after 5 days of incubation.

[0102] Temperatures before and directly after the processing chamber were measured using HYP-0 T-type thermocouples (Omega). Additionally, the maximum temperature was monitored indirectly using NTC resistors. Square-wave bipolar pulses, voltages, and currents within the processing chamber were recorded using a digital oscilloscope (Rigol DS1102E). Based on (Mastwijk, 2006) and (Timmermans et al., 2014), the electrical energy was obtained through numerical integration of the voltage and current trajectories and was equal to the calorific value within 10% of the experimental error.

[0103] All experiments were conducted in duplicate.

[0104] Table 2: PEF conditions used in this study

[0105] The test conditions are provided in Table 2. The dimensions of the treatment chamber were varied to obtain varying electric field strengths. Because the dimensions were variable, the residence time within the treatment chamber differed for each test condition. The frequency was adjusted to obtain the desired maximum temperature. Finally, the number of pulses was calculated by combining the residence time and frequency used. The time between two pulses was calculated by dividing the residence time by the number of pulses and subtracting the pulse duration.

[0106] Inactivation is expressed as the number of surviving microorganisms (N) under test conditions divided by the logarithm of the initial concentration of microorganisms (N0), i.e., log 10 (N / N0). In Figure 1A and Figure 1B As shown, inactivation is represented as varying with the highest temperature at the outlet of the treatment chamber of the test microorganism. The left image illustrates inactivation under various electric field strengths for short pulses (2 microseconds), representing the state of the art, while the right image illustrates inactivation under various electric field strengths for long pulses (i.e., 100 or 1000 microseconds), representing the invention described in this application.

[0107] exist Figure 1A and Figure 1B The image shows the variation of E. coli in orange juice with varying electric field strength and pulse duration. Escherichia coli Inactivation of ).

[0108] An increase in electric field strength at a constant pulse duration leads to greater inactivation at the selected highest temperature, which can... Figure 1A and Figure 1B Zhong Cong Figure 1A and Figure 1B The left image in the image (electric field strength of 10 kV / cm, 15 kV / cm, or 20 kV / cm; pulse duration of 2 microseconds) and Figure 1A and Figure 1B As can be seen in the right-hand image (electric field strength of 0.9 kV / cm or 2.7 kV / cm; pulse duration of 1000 microseconds).

[0109] for Figure 1A and Figure 1B Other microorganisms shown also exhibited the effect of this increased electric field strength, showing greater inactivation at higher electric field strengths. It is desirable that further increases in the electric field strength described in the conditions of this invention, up to 5 kV / cm, will further reduce the maximum temperature while still inactivating the desired number of microorganisms.

[0110] Unwilling to be bound by theory, this can be explained by the externally applied electric field (E) exceeding the critical electric field strength (Ec) of the membrane, causing further damage and inactivation (Alvarez). et al. (2006). Ec varies for different microorganisms, and it is generally agreed in this field that microbial inactivation requires an electric field strength greater than 5 kV / cm (Raso). et al. (2014). However, according to the present invention, the inventors now demonstrate for the first time that electric field strengths below 5 kV / cm, for example 2.7 kV / cm, are also effective for achieving microbial inactivation at sufficient levels of 1.0E-7 (N / N0) or lower. (Comparison) Figure 1A and Figure 1B As seen in the left and right images, an electric field strength of 2.7 kV / cm with a pulse duration of 100 microseconds or 1000 microseconds provides a higher degree of microbial inactivation compared to the higher electric field strength and shorter pulse duration typically used in this field.

[0111] To avoid being bound by theory, it is suggested that pulse duration plays a crucial role in the inactivation of microorganisms, implying an alternative mechanism to the conditions described in this invention, rather than the currently used one. It is hypothesized that when the pulse duration is sufficiently long, voltage-sensitive protein channels will open and conduct currents higher than they are designed to. As a result, these channels will become irreversibly denatured, and the cell will lose its viability.

[0112] Escherichia coli ( E. coli The inactivation data showed no difference in the degree of inactivation at 2.7 kV / cm when using pulses of 100 or 1000 microseconds, suggesting that the critical pulse duration is less than 100 microseconds.

[0113] Furthermore, this new PEF condition shows that Gram-positive and Gram-negative microorganisms can be inactivated up to 1.0E-7 (N / N0). Additionally, microbial size does not play a significant role in the degree of inactivation, whereas it does under current technological conditions. Although yeast at 2.7 kV / cm is more effective than *Lactobacillus plantarum* (… L. plantarum ) and Salmonella Sanftonburg ( S. Senftenberg bacteria can be inactivated at lower maximum temperatures, but no E. coli was found under the new PEF conditions. E. coli ) and Listeria monocytogenes ( L. monocytogenes There are significant differences between them, and using the currently used PEF conditions, a larger difference was found (left image).

[0114] Example 2: Escherichia coli in products with variable properties ( E. coli ) and Listeria monocytogenes bacteria( Listeria monocytogenes Microbial inactivation (1 L / h scale)

[0115] According to the description in (Timmermans) et al. The method in (2014) for Escherichia coli (E. coli) Escherichia coli(ATCC 35218) and Listeria monocytogenes ( Listeria monocytogenes NV8 was prepared from frozen primary culture and in tryptone soybean broth (E. coli ( Escherichia coli Or in brain and heart broth (Listeria monocytogenes) Listeria monocytogenes Cultivate in ))

[0116] After culturing, the cells were washed and suspended at different pH and conductivity (see...). Figure 2 Minute Maid orange juice ® A concentration of approximately 1.0E+8 cfu / mL was achieved in coconut water (Healthy People) or watermelon juice (freshly squeezed). The same PEF equipment system and settings as described in Example 1 were used, with a flow rate of 13.5 ± 0.5 mL / min. However, only one processing condition was used: an electric field strength of 2.7 kV / cm and a pulse duration of 1000 microseconds. Due to the different conductivity among the three products (… Figure 2 Therefore, different frequency settings are required, and the number of pulses required during treatment to achieve a similar maximum temperature for each product varies. Table 3 shows the range of test frequencies, number of pulses, and maximum temperature after treatment for the three test juices.

[0117] Inactivation is expressed as the number of surviving microorganisms (N) under test conditions, divided by the logarithm of the initial concentration of microorganisms (N0), i.e., log 10 (N / N0).

[0118] Table 3: PEF conditions used in this study

[0119] exist Figure 3A and Figure 3B The figure shows the levels of E. coli (E. coli) in various fruit juices at different maximum temperatures. Escherichia coli ) and Listeria monocytogenes ( Listeria monocytogenes Inactivation of E. coli ( ) was compared with that of various liquid matrices (i.e., liquid products orange juice, coconut water, and watermelon juice). Escherichia coli () Figure 3A ) and Listeria monocytogenes ( Listeria monocytogenes () Figure 3B When comparing the inactivation curves of the products (i.e., the liquid products orange juice, coconut water, and watermelon juice), it was observed that the inactivation initiation temperature was not different. Furthermore, the degree of inactivation was similar for orange juice, coconut water, and watermelon juice. This demonstrates that different pH values ​​and conductivity values ​​of the product matrix (i.e., liquid products orange juice, coconut water, and watermelon juice) did not affect the degree of inactivation. Additionally, it was found that for Gram-negative *Escherichia coli* (G. coli),... Escherichia coli(with bacterial cell size of approximately 0.7-1.5 micrometers x 2-5 micrometers) and for Gram-positive mononuclear cytoproliferative Listeria ( Listeria monocytogenes (with cell sizes of approximately 0.4–0.5 μm x 0.5–2 μm), and found that the inactivation curves at similar temperatures were comparable, indicating no difference for the microorganisms used.

[0120] This demonstrates that the method of the present invention, applied to orange juice, coconut water, and watermelon juice, is similarly effective and efficient in inactivating Gram-positive and Gram-negative bacteria in a variety of different liquid matrices, i.e., different liquid food products.

[0121] Example 3: Microbial validation at a scale of 1200 L / h

[0122] A batch of oranges (variety: Natal folha murcha) was commercially pressed to obtain 4,000 L of orange juice. The orange juice was not inoculated with microorganisms, so the microbial community naturally present in the orange juice was studied to validate the previously established processing conditions (1,200 L / h) at a large scale.

[0123] Orange juice was pumped at a flow rate of 1,200 ± 100 L / h and preheated to 59°C. Pulses were delivered in three vertically positioned processing chambers to achieve a maximum temperature of 70°C. The processing area of ​​the intermediate processing chamber (length: 14 mm, diameter: 8 mm) was smaller than that of the first and third (outer) processing chambers (length: 14 mm, diameter: 12 mm). Due to the power supply connection, this resulted in the electric field strength in the intermediate processing chamber being doubled compared to the outer processing chambers, i.e., the electric field strength in the intermediate processing chamber was 1.9 kV / cm, while the electric field strength in the outer processing chambers was 1.0 kV / cm. The pulses had a fixed duration of 1000 microseconds. The residence time in the intermediate processing chamber was 5.4 ± 0.5 ms and in the outer processing chamber it was 9.5 ± 0.9 ms, and the total number of pulses delivered during the processing at a repetition rate of 207 ± 18 Hz was 5.1 ± 0.01.

[0124] The juice is cooled directly within 3 seconds of leaving the processing chamber, minimizing holding time to reduce heat load on the product. After cooling, the juice is aseptically packaged and stored.

[0125] Microbial samples of untreated and PEF-treated juices were analyzed in duplicate in three different laboratories, resulting in a total of six untreated and PEF samples to be analyzed. Total thermophilic plate counts, total coliforms, and the number of yeasts and molds were analyzed according to the method described by Dowes and ITO (2001). Acidophilic thermosporogenic bacteria (ATSB) were analyzed according to the method of Eguchi et al. (1999), and Salmonella were analyzed according to the method of AOAC (2000). Salmonella ), according to the method of ISO 11290-1 (1996), analysis of Listeria monocytogenes ( Listeria monocytogenes Furthermore, the lactic acid bacteria were analyzed according to the method of Silva et al. (2007).

[0126] The results of microbial inactivation in untreated orange juice and PEF-treated orange juice are described in Figure 4A and 4B The initial microbial load in untreated orange juice is relatively high, as it is approximately 1.0E+5 cfu / mL. Figure 4A Following PEF treatment of the orange juice, no microorganisms were detected on the plates (total plate count), and no molds, yeasts, or coliforms were also detected on the plates (all plates showed <1 cfu / mL; see [link to article]). Figure 4A ).exist Figure 4B In the study, qualitative data showed that all lactic acid bacteria and acidophilic thermospore-forming bacteria (ATSB) present in untreated orange juice were inactivated during the PEF treatment of the orange juice.

[0127] Example 4: The effect of the PEF conditions of the present invention on quality and microbiological shelf life

[0128] During the 3-month shelf life, samples of PEF-treated orange juice produced and described in Example 3 were analyzed weekly. Quality aspects and microbial counts were analyzed during this period. Microbial analysis was performed using methods similar to those described in Example 3. For quality analysis, the amount of soluble solids (JBT FoodTech Citrus Systems, 2011-1), acidity (JBT FoodTech Citrus Systems, 2011-2), pH (JBT FoodTech Citrus Systems, 2011-3), oil content (JBT FoodTech Citrus Systems, 2011-4), vitamin C content (JBT FoodTech Citrus Systems, 2011-5), and pectin esterase activity (Rouse and Atkins, 1955) were measured at indicated times according to methods known in the art. Samples were stored at 7°C and at room temperature (ambient temperature 20-25°C) to facilitate accelerated shelf-life studies.

[0129] Microbiological evaluation of the samples was performed, and the results are shown in Table 2. Throughout the entire shelf life of 104 days, the microbial counts of the PEF-treated samples showed less than the detection limit of 1 cfu / mL when the orange juice was stored at 7°C and when it was stored at ambient temperature.

[0130] Table 4. Results of microbiological analysis of PEF-treated orange juice stored at 7°C or at ambient temperature during its shelf life.

[0131]

[0132] NA. Unanalyzed

[0133] Sensory evaluations were conducted by the training team. The samples were evaluated for their overall appearance, color, odor, and taste. Samples were rated "good" when their characteristics were similar to freshly squeezed orange juice, and "bad" when they no longer possessed the superior qualities of freshly squeezed juice.

[0134] exist Figure 5A and Figure 5B Sensory analysis results are shown during the shelf life. Samples stored at 7°C showed similarities to freshly squeezed orange juice up to a shelf life of 64 days. After this storage period, the fresh aroma and flavor diminished, although the appearance and color of the juice were considered "good" until 90 days of storage at 7°C. Figure 5A PEF-treated juice stored at ambient temperature is only acceptable up to 27 days after storage. Figure 5B After this time, it can no longer be considered fresh juice.

[0135] The quality of the orange juice was also monitored. Untreated juice was evaluated prior to PEF treatment to assess the impact of this method on these aspects.

[0136] Total soluble solids (°Brix) Figure 6 The curves for experiments conducted at 7°C and at ambient temperature overlap in the graph for 6 days and longer), acidity ( Figure 7 pH Figure 8 ), oil content ( Figure 9 ) and vitamin C content ( Figure 10 The properties remained unchanged due to PEF treatment and did not change during storage at 7°C or at ambient temperature.

[0137] Differences in pectin methyl esterase (PME) activity were observed before and after PEF treatment. Figure 11 Due to the temperatures generated during PEF processing, some PME enzymes are inactivated, resulting in reduced enzyme activity. That is, the heat generated during processing according to the invention raises the juice temperature to 70°C. Juice processed with PEF according to the invention is preheated to 59°C before being introduced into a continuous-flow PEF apparatus. This enzyme inactivation is one of the goals of pasteurization, as residual enzyme activity can cause the citrus juice to lose its turbidity and gel during storage. The enzyme activity is expressed as the release of acid per mL (multiplied by 1.0E+4) over time during pectin hydrolysis at pH 7.8 and 20°C. Typically, most juice pasteurizers provide juice with pectin esterase activity values ​​between 1.0E-6 and 1.0E-4, expressed in pectin esterase units (PEU).

[0138] References

[0139] - AOAC (2000). AOAC Official Method 989.13-1998. Motile Salmonella inall foods. AOAC International, Official Method of Analysis of AOACInternational.

[0140] - Álvarez, I., Condón, S., Raso, J., (2006). Microbial inactivationby pulsed electric fields. In: Raso, J., Heinz, V. (Eds). Pulsed ElectricFields Technology for the Food Industry, Fundamentals and Applications.Springer, New York.

[0141] - Bergey, 1986. Manual of systematic bacteriology. Williams andWilkins, Baltimore

[0142] - Campos, F.P., Cristianini, M. (2007) Inactivation of Saccharomyces cerevisiae , and Lactobacillus plantarum in orange juice using ultra high-pressure pasteurisation. Innovative Food Science and Emerging Technologies,8, 226-229

[0143] - Doyle, M.E., Mazotta, A.S.(2000) Review studies on the thermalresistance of Salmonella . Journal of Food Protection, 63, 779-795

[0144] - Dowes, F.P., Ito, K., (2001). Compendium of methods for themicrobiological examination of foods. 4 th edition. American Public HealthAssociation, Washington, D C.

[0145] - Eguchi, S.Y., Manfio, G.P., Pinhatti, M.E., Azuma, E., Variane,S.F. (1999). Acidothermophilic sporeforming bacteria (ATSB) in orange juices:detection methods, ecology, and involvement in the deterioration of fruitjuices. Report of the research project ABE Citrus, Campinas (SP), Brasil.

[0146] - El Zakhem, H., Lanoisellé, J-L., Lebovka, N.I., Nonus, M.,Vorobiev, E. (2006) Behavior of yeast cells in aqueous suspension affected bypulsed electric field. Journal of Colloid and Interface Science, 300, 553-563

[0147] - El Zakhem, H., Lanoisellé, J-L., Lebovka, N.I., Nonus, M.,Vorobiev, E. (2007) Influence of temperature and surfactant on Escherichia coli inactivation in aqueous suspensions treated by moderate pulsed electricfields. International Journal of Food Microbiology, 120, 259-265

[0148] - Eynard, N., Rodriguez, F., Trotard, J., Teissié, J. (1998)Electrooptics studies of Escherichia colielectropulsation: orientation,permeabilization and gene transfer. Biophysical Journal, 75, 2587-2596

[0149] - Gurtler, J.B., Bailey, R.B., Geveke, D.J., Zhang, H.Q. (2011)Pulsed electric field inactivation of E. coli O157:H7 and non-pathogenicsurrogate E. coli in strawberry juice as influenced by sodium benzoate,potassium sorbate, and citric acid. Food Control, 22, 1689-1694

[0150] - Heinz, V., Alvarez, I., Angersbach, A., Knorr, D. (2002).Preservation of liquid foods by high intensity pulsed electric fields - basicconcepts for process design. Trends in Food Science & Technology, 12, 103-111

[0151] - ISO 11290-1 (1996). Microbiology of food and animal feeding stuffs.Horizontal method for the detection and enumeration of Listeria monocytogenes- Part 1: detection method.

[0152] - JBT Food Tech Citrus Systems (2011-1). Total soluble solids byrefractometer. In: Procedures for analysis of citrus products. 6th Edition,Lakeland.

[0153] - JBT Food Tech Citrus Systems (2011-2). Total titratable acidity(industry method) In: Procedures for analysis of citrus products. 6 th Edition,Lakeland.

[0154] - JBT Food Tech Citrus Systems (2011-3). pH. In: Procedures foranalysis of citrus products. 6 th Edition, Lakeland.

[0155] - JBT Food Tech Citrus Systems (2011-4).Recoverable oil (Scottmethod). In: Procedures for analysis of citrus products. 6 th Edition,Lakeland.

[0156] - JBT Food Tech Citrus Systems (2011-5). Ascorbic acid by Iodinetitration. In: Procedures for analysis of citrus products. 6 th Edition,Lakeland.

[0157] - Loeffler, M.J. (2006) Generation and application of high intensitypulsed electric fields. In: Heinz, V., Raso, J. (Eds.) Pulsed electric fieldtechnology for the food industry - fundamentals and applications. Springer,New York.

[0158] - Mastwijk, H.C. (2006) Pulsed Power systems for application ofpulsed electric fields in the food industry. In: Heinz, V., Raso, J. (Eds.)Pulsed electric field technology for the food industry - fundamentals andapplications. Springer, New York.

[0159] - Mastwijk, H.C., Gulfo-van Beusekom, K., Pol-Hofstad, I.E., Schuten,H., Boonman, M, Bartels, P.V. (2007). Definitions and guidelines forreporting on pulsed electric field experiments. In: Lelieveld, H.L.M.,Notermans, S., de Haan, S.W.H. (Eds.). Food preservation by pulsed electricfields. From research to application. Woodhead publishing.

[0160] - Raso, J., Condón, S., Álvarez, I., (2014). Non-thermal processing.Pulsed Electric Field. In: Batt, C.A. (Ed). Encyclopedia of Food Microbiology(second edition). Elsevier.

[0161] - Put, H.M.C., De Jong, J., Sand, F.E.M.J., Van Grinsven, A.M. (1976)Heat resistance studies on yeast spp. causing spoilage in soft drinks.Journal of Applied Bacteriology, 40, 135-152

[0162] - Rouse, A.H., and Atkins, C.D. (1955) Pectinesterase and pectin incommercial citrus juices as determined by methods used at the citrusexperiment station. Florida Agricultural Experiment Stations, 570, 3-19.

[0163] - Saulis, G., Wouters, P.C. (2007). Probable mechanisms ofmicroorganism inactivation by pulsed electric fields. In: Lelieveld, H.L.M.,Notermans, S., de Haan, S.W.H. (Eds.). Food preservation by pulsed electricfields. From research to application. Woodhead publishing.

[0164] - Sharma, P., Bremer, P., Oey, I., Everett, D.W. (2014) Bacterialinactivation in whole milk using pulsed electric field processing.International Dairy Journal, 35, 49-56

[0165] - Silva, N., Junqueira, V.C.A., Silveira, N.F.A., Taniwaki, M.H.,Santos, R.F.S., Gomes, R.A.R., Okazaki, M.M., ( 2007). Manual de Métodos deanálise microbiológica de alimentos, third edition, São Paulo.

[0166] - Timmermans, R.A.H., Nierop Groot, M.N., Nederhoff , A.L., vanBoekel, M.A.J.S., Matser, A.M. Mastwijk, H.C. (2014). Pulsed electric fieldprocessing of different fruit juices: Impact of pH and temperature oninactivation of spoilage and pathogenic micro-organisms. InternationalJournal of Food Microbiology, 173 , 105-111.

[0167] - Tsong, T.Y. (1992) Time sequence of molecular events inelectroporation. In: Chang, D.C., Chassy, B.M., Saunders, J.A., Sowers, A.E.(Eds). Guide to electroporation and electrofusion. Academic Press

[0168] - Van der Veen, S., Wagendorp, A., Abee, T., Wells-Bennink, M.H.(2009). Diversity assessment of heat resistance of Listeria monocytogenesstrains in a continuous-flow heating system. Journal of Food Protection, 5,999-1004

[0169] - Wouters, P.C., Dutreux, N., Smelt, J.P.P.M., Lelieveld, H.L.M.(1999). Effects of pulsed electric fields on inactivation kinetics of Listeria innocua . Applied and Environmental Microbiology, 65, 5364-5371。

Claims

1. A method for rapidly and uniformly heating a liquid product to a predetermined temperature using resistance heating to obtain a heated liquid product, comprising: (a) Providing liquid products; (b) Provide an apparatus for rapidly and uniformly heating a liquid product to a predetermined temperature using a pulsed electric field via resistance heating; (c) Continuously supplying the liquid product to the inlet of the device and allowing the liquid product to flow through the device; (d) Frequently generating pulsed currents for the liquid product flowing through the device, wherein at least one pulse is applied to each fluid element during passage, the pulse duration is 100 to 1000 microseconds, and the electric field strength is 0.9 to 2.7 kV / cm; and During the resistance heating, the maximum temperature of the liquid product is autonomously maintained between 40°C and 85°C. The method described therein is a method for inactivating microorganisms in the liquid product. The microbial count (CFU) in the liquid product is reduced by at least 5 log CFU / mL.

2. The method according to claim 1, wherein the pH of the liquid product is between pH 1.5 and pH 9.

0.

3. The method according to claim 1, wherein the conductivity of the liquid product measured at 20°C is between 0.1 and 3 S / m.

4. The method according to claim 1, wherein the liquid product is a liquid food product or a liquid feed product.

5. The method according to claim 4, wherein the liquid product is a raw material, a semi-finished product, or a final liquid product, such as fruit juice, vegetable juice or smoothie, jam, spread product, alcoholic or non-alcoholic beverage, dairy product, plant-based milk product, liquid egg, soup, or sauce.

6. The method of claim 5, wherein the dairy product is selected from milk, dairy products, or liquid compositions comprising milk components or milk fractions.

7. The method of claim 1, wherein the temperature of the liquid product is autonomously maintained below 75°C.

8. The method according to claim 1, wherein the electric field strength is 2.5 to 2.7 kV / cm.

9. The method of claim 1, wherein a bipolar pulse is applied.

10. The method of claim 1, wherein the heated liquid product is immediately cooled after flowing through the device.

11. The method of claim 1, wherein the liquid product is preheated to a temperature ranging from 20°C to 70°C before being supplied to the device.

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