Inactivating Food Microbes by High-Pressure Processing and Combined Nonthermal and Thermal Treatment: A Review

Department of Food Engineering, National Institute of Food Technology Entrepreneurship and Management, Kundli, Sonepat 131028, Haryana, India Department of Food Process Engineering, National Institute of Technology Rourkela, Odisha 769008, India Department of Food Technology, Rajalakshmi Engineering College, Chennai-602105, Tamil Nadu, India Department of Food Technology, School of Chemical Technology, Harcourt Butler Technical University, Kanpur 208002, Uttar Pradesh, India Livestock Production and Management Section, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, Uttar Pradesh, India


Introduction
Most food commodities, i.e., fruits, vegetables, meat, poultry, seafood, milk, and their products, are perishable due to limited shelf life. e presence of moisture and environmental conditions (temperature and relative humidity) around food products during storage triggers physical, chemical changes, and microbiological growths leads to food deterioration or spoilage [1]. Spoilage can be defined as undesirable changes that render a product unsuitable for consumption due to physical, chemical, or microbiological changes. Physical changes include loss of moisture from dried foods, gain of moisture, and freeze burn. In many cases, physical changes in food products during storage also lead to chemical reactions and microorganism growth. Some undesirable chemical changes are staling, discolorations (by enzymatic browning and nonenzymatic browning), offflavor development (due to oxidation of food leading to rancidity), etc. in the food products [2]. ese chemical changes or reactions can be triggered by specific enzymes, i.e., lipases, peroxidases, polyphenol oxidases, and catalases. ese changes reduce food quality and acceptability by consumers and are not recommended for consumption. Microbial spoilage can occur when bacteria, mold, and yeast grow in food or produce toxins harmful to humans. Apart from storage temperature and relative humidity, the growth of microorganisms in the food also depends on food composition. Since food is a rich source of nutrients and water activity of most of the fresh commodities is high. So, it provides a very suitable environment for the microorganism to thrive on food. Food deterioration by the growth of pathogenic microorganisms is a significant concern [2]. As pathogenic bacteria are linked with outbreaks of fruits, vegetables, dairy, meat, and poultry-based products. Taiwan Food Drug Administration reported more than 2000 cases of food poisoning due to accidental consumption of fresh fruits, vegetables, and seafood products contaminated with pathogenic microbes [3]. So, it is important to process food commodities to keep them safe and as well as to extend their shelf life.
Different processing techniques (i.e., thermal and nonthermal technologies) are used to prevent physical contaminations, slow down the chemical, enzymatic reactions, and eliminate/reduce microbial spoilage. Although thermal processing (which involves applying heat) is the most used and is a well-established treatment in terms of its historical use, predictability, and cost, it impacts the nutritional quality due to the high temperature involved while processing. Nowadays, nonthermal techniques (which involve applications of pressure, short pulse electric field, light, and sound waves) are preferred over thermal techniques. As the temperature attained by the product during nonthermal processing is low, lower nutritional losses occur during food processing [4]. Moreover, consumers prefer minimally processed food with a clean label and products processed with nonthermal techniques. High-pressure processing (HPP), pulse electric field, irradiation, cold plasma, and ultrasound are some of the nonthermal processing techniques. Among these nonthermal techniques, HPP in compliance with consumer requirements, provide products similar to fresh, clean label, additives-free, and convenient with extended shelf life. It is a promising cold pasteurization technology and gaining importance worldwide [5][6][7].
For commercial processing, HPP utilizes the application of high pressure (100-600 MPa) for a particular time at room temperature to packaged food kept inside a vessel to inactivate microorganisms. e vessel contains solvent (i.e., water, propanol ethanol, etc.), which transmits the pressure equally and uniformly throughout the vessel [8]. HPP of food commodities leads to changes in the cell membrane, cell morphology, biochemical reactions, and alteration in the genetic mechanism responsible for microorganism inactivation. ese effects vary with the type of microorganism and food composition. Hence, it is of utmost importance to optimize the process parameters (pressure/holding time/ temperature) to ensure food safety with adequate margins [9].
Food with low pH values (pH values < 4.6) indicating high acid intensity is less likely to be spoiled by bacteria. But, low acid foods (pH values > 4.6) are less microbiologically stable, and bacteria tend to produce a dormant form known as spores in fresh food [10]. Spores are tough to kill/inactivate, and if they are not appropriately inactivated, they wait for favorable conditions to grow [11]. So, it is of utmost importance to kill spores. HPP at room temperature is not adequate to inactivate all the spores, especially in the case of low-acid foods. To achieve a higher efficacy in inactivating pressure-resistant pathogens and spores, temperature (90-120°C) can simultaneously be increased during HPP. e combination of heat (90-120°C) with pressure sequentially or simultaneously has been reported to provide synergistic effects against spores on different food commodities [12]. Similarly, sequential use of other techniques like irradiation, preservatives along with HPP has shown synergistic and additive effects against spores/pathogens to achieve food commodities safety [13][14][15].
Keeping food safety in view, the main objective of this review is to summarize the existing data from the published research concerning the effects of high-pressure treatment to inactivate microorganisms in fruits and vegetables; milk and milk products; meat, poultry, and seafood, and their products. Sometimes, high pressure alone is ineffective for the complete inactivation of pathogens and spores; therefore, a different strategic combination of thermal and nonthermal techniques assisting high pressure is also assayed. e provided information would be beneficial to interested researchers and industry personnel.

High-Pressure Inactivation in Specific
Food Sector e effectiveness of treatment to inactivate microbial populations depends on the type of microorganism, species, types of food (plant or animal origin), and matrix of food. e lethal effect of HPP on microbial population is assumed to be due to simultaneous effects on cell membrane permeability, changes in cell morphology, altered biochemical reactions, interference in the genetic mechanism, which occurs in the cell of microorganism, and detailed mechanism has been reported by Sehrawat et al. [16].
2.1. Fruits, Vegetables, and eir Products. Fresh agricultural products are healthy and nutritious, but contamination by microorganisms has been reported during storage. Second, there is a huge demand for refrigerated stored salad, freshcut fruits, and vegetables available in the market. Apart from being healthy, the availability of these products from the market saves time and provides convenience to the customers. But during cutting and packaging, chances of growth of E. coli O157: H7 and Salmonella might occur. Recently, frequent food-borne pathogens outbreaks are linked with these products and are of major public health concern. Contamination of raw products with pathogenic microorganisms can be due to their direct growth or indirect sources such as insects, water, and soil. HPP has been proven to be an effective technology for eliminating these pathogens of concern [17]. e effectiveness of HPP in reducing the microbial population is given in Table 1.
Apart from providing microbial safety, another reason for considering HPP as an alternative to convention preservation techniques is its limited effects on covalent bonds resulting in an only minor modifications in nutritional and sensory aspects. Pressure-treated juices are now available on a commercial scale in many countries viz. France  [46] (Ultrifruit), Japan (Waka Food Industries), Portugal (Frabaca), the UK (Orchard House), the USA (Odwala), and Mexico (Grupo Jumex). Different fruits and vegetables that have been processed using high-pressure are apples, litchi, orange, papaya, pomegranate, kiwi, plum, pineapple, cashew apple, green beans, cabbage, radish, carrot, spinach, wheat grass, onion, etc.
Different microflorae require different pressure treatments in order to inactivate them. Pathogenic E. coli was the most reported to be a more resistant bacterial strain than Listeria monocytogenes in mango juice to high-pressure treatment [41]. Around 6 log reduction of E. coli O157: H7 and 5 log reduction of L. monocytogenes were achieved at 400 MPa for 10 min and 500 MPa for 1 min; there were no survivors of E. coli. For Z. bailii, P. membranaefaciens, and L. mesenteroides, the pressure of 300 MPa was sufficient to reduce the count to less than 1 log CFU/mL [41]. So, the most resistant microorganism can be selected as the target microorganism to get the optimum conditions for the treatment. Pilavtepe-Çelik et al. [48] reported the inactivation of E. coli O157: H7 and Staphylococcus aureus by high-pressure treatment  MPa/0-40 min/40°C) in carrot juice and peptone water. e carrot juice medium showed pressure resistance to E. coli (add pressure treatment), whereas S. aureus (add pressure treatment) was more resistant to peptone water than the carrot juice medium. is specific effect on S. aureus is due to the release of naturally occurring constituents of phytoalexins (6-Methoxymellein from carrot root, which is an antimicrobial compound produced with the response to microbial infection) in cellular and vascular fluids, exerting a toxic effect. It was also evident from earlier literature that this 6-methoxymellein from carrot cells was more effective against Gram-positive bacteria than Gram-negative bacteria [54]. So, it is important to consider the medium in which treatment is given as it can have varied results. Another study on carrot juice pressurization at 500 and 600 MPa for 1 min at 20°C showed a significant reduction in microbial count from (4 log reduction), and a shelf life of 22 days was reported at 4°C [49]. During the storage study, at 8°C count was higher, although it took a long time to reach the maximum level, it was lower than the control samples. Pressure treatment of carrot juice at 500 MPa/1 min/20°C followed by storage at 8°C, for 22 days inactivated the competitive microflora except for spore formers and L. lactis (non-spore former) [49]. It can be concluded that apart from the amount of pressure, duration of treatment, and storage temperature; the type of microorganism plays an important role. e juice of wheatgrass was given different treatments, i.e., thermal (75°C/15 s), HPP (500 MPa/60 s), and ultraviolet-C light (254 nm/69.2 mJ/cm 2 ) to achieve 5 log CFU reduction of microorganisms. Although all the treatment conditions mentioned above were found to be effective in the inactivation of microorganisms like E. coli P36, L. innocua ATCC 51742, and S. typhimurium WG49. However, thermal processing leads to a reduction in chlorophyll content, antioxidant properties, and loss of color [50]. So, HPP was reported to be the preferred method of processing as it retained maximum nutrients and gave a higher yield, and was recommended for other beverages with the same equivalent treatments. Apart from the safety of food, quality is important and is given diligent consideration by processors and consumers. Similarly, in another study by Chang et al. [44]; HPP and thermal treatment were given to white grape juice. ermal processing (90°C/60 s) and HPP (600 MPa/3 min) were found to be effective in increasing the shelf life of white grape juice for 20 days. Differences in HPP processed juice and fresh were not significant based on sensory analysis, but thermally processed juice showed low acceptance [44]. e initial population of aerobic plate count, Y&M, and coliform count for control juice were 3.2, 2.2, and 2.1 log CFU/mL. When compared to treatment at 300 MPa/3 min, 600 MPa/3 min showed a significant  [44]. HPP treatment (600 MPa/5 min/35°C) of elephant apple juice extended the shelf life of juice by 60 days at 4°C (microorganism count was <1 CFU/mL), whereas the microbial count was higher in thermally processed and control samples during the shelf-life study. e untreated samples showed a continuous increase in microorganism number during the storage study and were unacceptable by the end of 10 days as total viable bacteria; Y&M were 6.23 and 4.06 CFU/mL, respectively [45].
For pressure treatment (400 MPa/10 min, 500 MPa/ 5 min, and 600 MPa/2.5 min) low acid foods like sweet potatoes reduced Y&M to below detection levels where the initial count was 6.06 log10 CFU/mL. Further, Y&M was not detected for up to 84 days when samples were stored at 4 and 25°C. However, better quality was reported in samples stored at 4°C, indicating the importance of storage temperature [52]. Similarly, in cantaloupe puree, pressure treatment of 400-500 MPa for 5 min drastically reduced Y&M, and no regrowth was observed up to 10 days of storage at 4°C [31]. A comparative effect of sustained pressure treatment, pressure pulses, and pressure cycles was done on pineapple juice and nectar inoculated with B. nivea. It was concluded that at 600 MPa pressure, the effect of cycles was more effective in B. nivea ascospore inactivation than treatment under sustained high pressure. In addition to ascospores, Y&M counts were also reduced to below detection levels [55].
Y&M spores are readily inactivated at 400 MPa except for certain ascospores of heat-resistant mold such as Byssochlamys nivea, Neosartorya fischeri, and Talaromyces macrosporus. [56]. In general, these ascospores are often associated with spoilage of pasteurized fruit products also, such as juice, jams, purees, and candied fruits. Besides, their presence in processed food may cause deleterious effects due to the production of mycotoxins. Santos et al. [57] identified twelve highly resistant mold species, including Neosartorya fumigate (23.6%), N. fischeri (19.1%), and Byssochlamys nivea (5.5%) being the predominant species in high acid pasteurized fruit products such as strawberry puree, orange juice, and apple puree. e resistance of these ascospores depends on the spore age and species. e older the spore higher its resistance to processing.
HPP has been successfully applied for the effective inactivation of different pathogens in various fruits, vegetables, and their products. e amount of pressure and time required to inactivate the microorganisms depends on the food category. Optimized process parameters conditions for one product cannot be generalized for all the products. Among the different factors that plays important role in achieving microorganism inactivation are type and age. As bacteria, Y&M against pressure offers varying resistance. Combination treatment is reported to be more effective against spores.

Milk and Milk Products.
Treating milk by high-pressure breaks only ionic and hydrophobic bonds of macromolecules (proteins) but does not denature bioactive proteins present in it. Very little or no effect on small molecules of milk components (vitamins, flavor, and amino acids) color, and other nutritional components have been reported along with effective microbial inactivation [58]. Other desirable changes induced are denaturation, gelling, and aggregation of proteins, which also influence the yield of dairy products produced from treated milk. Various researchers have successfully treated milk [59] and milk products like cheddar cheeses [60], gorgonzola cheese [61], and Queso Fresco cheese [62] using HPP for extended shelf life ( Table 2).
Raw milk acts as a carrier for the transmission of bacteria like E. coli, Salmonella, shigella, and S. aureus. ese microorganisms become part of untreated milk while milking milk from animals in barnyards, transporting milk, and storing milk at chilling centers. ese food-borne pathogens are of public health concern. Yang et al. [66] worked on the inactivation of these bacteria in milk by HPP treatment. e duration of pressure treatment for 20, 30, 40, and 50 min at 300 MPa exhibited the highest inactivation rate of Salmonella and the lowest inactivation rate of S. aureus. e satisfactory duration for milk treatment was optimized to be 30 min. With an increase in pressure from 100 to 200 MPa, an increase in inactivation rates was observed. e inactivation was slower for Salmonella and E. coli and rapid for Shigella and S. aureus. It was concluded that a pressure of 300 MPa for 30 min at 25°C was sufficient to cause bacterial inactivation in milk. Most resistant S. aureus must be considered an indicator bacterium in milk when HPP was employed as a preservation technique. Efficacy of HPP in the destruction of Mycobacterium avium ssp. Paratuberculosis in milk was done by Donaghy et al. [64]. Pressure at 500 MPa for 10 min resulted in a 6.52 log reduction of the target microorganism.
A study was conducted by Narisawa et al. [72] to assess the injury and inactivation of Escherichia coli K-12 in different mediums, i.e., skimmed milk and its protein fractions (casein, whey, globulin, and albumin) by HPP treatment. It was revealed that skimmed milk had the most remarkable protective effect on inactivation. Moreover, the shielding effect was enhanced with an increase in the concentration of skimmed milk [72]. e presence of casein and lactose in milk also shields bacteria in milk during HPP [73]. e divalent cations Ca 2+ and Mg 2+ also shield bacteria against high-pressure-induced inactivation due to their stabilizing effect over the cellular membrane [16]. So, it is important to know the medium composition to optimize the pressure effective in overcoming the shielding effect provided to microorganisms by food.
Evidence of the repair mechanism of injured microbes in food, especially for low acid foods, has been reported, questioning the microbiological safety of foodstuffs.
Mechanism of repair after injury of most pressure-resistant strains of two Gram-positive (L. monocytogenes CA and S. aureus 485) and Gram-negative bacteria (E. coli O157:H7 933 and S. enteritidis FDA) inoculated in milk, was studied [74]. Inoculated milk was given HPP treatment (350-550 MPa) and was stored at 4, 22, and 30°C. ree stages of microbes after pressure treatment were established: i.e., (i). Cells can form visible colonies plated in both selective and nonselective agar called active cells (AC), (ii). Cells that undergo structural injury like cell wall/cell membrane injury and can form colonies only on nonselective agar are called I1 injury or primary injury, (iii). Cells that undergo metabolic injury cannot form colonies in both selective and nonselective agar are called I2 injury or secondary injury. However, in the repair of I2 injury, cells can form colonies on nonselective agar, similar to I1 injury. Except for L. monocytogenes CA, other bacteria were inactivated or injured in milk at 350 MPa. S. aureus cells in milk after pressure treatment at 350 and 450 MPa observed on day 1 after storage at 22 and 30°C showed I1 type injury. Whereas pressure-treated milk samples at 350 MPa, after 1 day of storage, caused E. coli cells to repair from I1 state to active cell. erefore, storage temperature and duration can alter the repair of bacterial cells, thereby influencing microbiological safety. Studies suggest that after the HPP of food, immediately injured cells might not be present but can recover during storage. So, a strategic combination with other processing techniques might effectively prevent the recovery of injured cells.
Yogurt is a fermented beverage prepared from milk in cooperation with two homofermentative bacteria Streptococcus thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus.
e excess lactic acid bacteria in yogurt have attributed beneficial effects; nonetheless, the post-acidification during the cold chain and modification of viable lactic acid bacteria count are bottlenecks. ermal processing after fermentation, in this case, would not be a viable solution to preserve the lactic acid bacteria at desired levels, and therefore novel technique like HPP plays a role. e effect of high-pressure treatment on the microflora of yogurt was investigated by Jankowska et al. [70]. ey found that highpressure treatment at 500 MPa does not significantly decrease the inactivation rate. In contrast, pressure treatment at 600 MPa/15 min showed a significant increase in bacterial inactivation from the initial load of ∼10 8 -10 9 CFU/mL to ∼10 2 -10 3 CFU/mL. It was found that Streptococcus thermophiles were slightly more resistant to high pressure than L. bulgaricus. Besides, yogurt was found to maintain acidity throughout the storage period after HPP treatment as it sufficiently reduced the acidifying bacteria. It was concluded that pressure treatment of 550 MPa for 15 min was optimum for yogurt processing with good sensory and textural characteristics with a shelf life of 4 weeks at 4°C. Microbial survivability in yogurt depended on the initial bacterial load and acidity of the sample [70]. e development of uniformly consistent microstructure in probiotic yogurt with improved gel strength and viscosity was accomplished by treating milk with HPP before fermentation. e development of uniformly consistent microstructure in probiotic yogurt with improved gel strength and viscosity was accomplished by treating milk with HPP before fermentation [75]. Cheese is a fermented dairy product in wide demand all over the world. Improved characteristics of cheese were reported after HPP. Studies showed that high pressure imparts the following: (i) e alters the proteolytic activity of cheese [76], (ii) Improve the softness of cheese [77], (iii) Affect the rennet coagulation of milk [76], (iv) Increase the shelf life [76], (v). Increase the cheese yield [76], and (vi) Improve the physicochemical properties of soft cheese [78]. In corresponding to microbial inactivation of cheese under high pressure, several studies have shown promising results in improving shelf life without affecting its inherent quality. e effect of high-dynamic pressure on different types of milk and its effect on the quality of the cheese was studied by Kheadr et al. [60]. ey found that 3-4 log reduction in L. innocua and 2-4 log reduction in total viable bacteria count was achieved by pressurizing milk, specifically the reduction in the microbial count was higher in low-fat milk. e reason is that milk fat acts as a protective medium for bacteria under high dynamic pressure, thereby preventing its destruction [60]. us, applying high-pressure to skim milk or low-fat milk employed for cheese preparation resulted in cheese being firm, cohesive, less brittle, and compact protein matrix with satisfactory microbiological quality. e cheeses prepared from low-fat pressurized milk show an initial listeria count of 10 6 CFU/mL was decreased to 10 2 CFU/mL after 3 months of ripening. Delgado et al. [69] reported that HPP at 400 and 600 MPa for 7 min of raw goat milk cheese resulted in inactivation of Mesophilic, aerobic, Enterobacteriaceae, Lactic acid bacteria, and Listeria spp., and differences in texture were observed. But the differences in control and pressure-treated samples were not observed by trained panelists and consumers. López-Pedemonte et al. [68] investigated the effect of ultrahigh pressure homogenization (UHPH) and HHP processing on the inactivation of S. aureus CECT 976 in milk to be employed for cheese making. e UHPH was employed at 300 and 30 MPa at primary and secondary homogenization stage, resp., followed by HHP treatment at 400 MPa/10 min/20°C. ey found that S. aureus was present in cheese initially at a load of 8.5 log10 CFU/g in control. After UHPH and HHP treatment of milk, the cheese after 15 days of ripening showed complete inactivation of S. aureus and its enterotoxin.
In general, flavor, color, and nutrients were significantly retained after the pressure treatment of milk and its products. However, to prevent the recovery of injured cells during storage, a strategic combination of HPP with other thermal and nonthermal treatments can be looked upon.

Meat, Poultry
, and Seafood. Meat, poultry, and seafood are high in moisture content and rich in protein and thus, these products have been associated with frequent outbreaks of food poisoning and food-borne diseases. Major outbreaks were linked with dog meat in China [79], red meat caused infectious intestinal infection disease outbreaks in the United Kingdom [80], and multiple outbreaks were due to frozen oyster in Australia [81]. A survey by FDA reported the presence of L. monocytogenes in cold-smoked salmon with a 17% frequency and 4% incidence in hot smoked fish and shellfish [82]. In Europe, 191 cases of death due to the eating of crustaceans and shellfish in which the presence of Listeria was reported in the year 2013 [83]. Listeriosis outbreak has been increasing in Europe for the last few years, with a fatality rate of 13.8% in 2017 EFSA (European Food Safety Authority) and ECDC (European Centre for Disease Prevention and Control) [84]. ese implications could be minimized by using HPP, which also simultaneously retains the natural aroma, appearance, flavor, texture, and nutrient value of the products [85,86]. e effect of high-pressure processing on deboned drycured hams was investigated by Perez-Baltar et al. [87]. ey found that high pressure of 600 MPa for 5 min inactivated L. monocytogenes at the surface during 60 days of storage at 4 and 12°C. However, variation in the moisture content, water activity, and salt and nitrate content on the surface and interior of dry-cured ham showed variation in pathogen inactivation. Around 2 log reduction in surface and 3 log reduction in the interior of dry-cured hams were accomplished during HPP treatment. As per USDA and European criteria, food safety against L. monocytogenes could be attained by HPP treatment at 600 MPa/5 min. A study on the inactivation of Shiga Toxin-Producing Escherichia coli (STPE) by HPP treatment (400-600 MPa/0-18 min) was studied by Porto-Fett et al. [6]. Major conclusions drawn from the study were that refrigerated and frozen storage of meatballs prior to HPP resulted in similar pathogen reduction, i.e., 0.9 to 2.9 log CFU/g at 4°C and 1 to 3 log CFU/g −20°C. Only 1-3 min were required at 600 MPa as compared to 9 min at 400 MPa to achieve a ≥ 2.0 log CFU/g. In another study, HPP treatment significantly reduced microbes in pork burgers [88]. e addition of 2% rice bran extract followed by HPP treatment at 600 MPa/5 min did not significantly affect microbial reduction but improved the quality of the pork burger. Rice bran extract acted as a natural antioxidant in maintaining the stability of burgers during refrigerated storage. Nevertheless, in comparison to rice bran extract, HPP treatment was effective in microbial inactivation and in extending the shelf life of pork burgers up to 21 days. Bonilauri et al. [89] reported the effect of the processing method and HPP on reducing Salmonella Spp. in Italian salami production. From the 20 different samples of salami, they identified a significant relationship between salmonella reduction and process parameters time/temperature of acidification/drying, time/ temperature of seasoning, pH, and aw) during sausage production. e management of sausage production process parameters decreased the salmonella load by 0.97-4.67 Log CFU/g but was insufficient to achieve 5 log reduction requirements of export to the USA, whereas the additional hurdle in the form of HPP treatment resulted in 2.41-5.84 Log CFU/g. e study aimed to identify and implement appropriate HACCP plans to control the risk of salmonella in Italian Salami using the appropriate HPP technique.

Journal of Food Quality
Meat, poultry, and fish are perishable products with limited shelf life, and microorganisms easily thrive over them during various stages such as cutting, mincing, protein solubilizing with salt, product forming, or packaging [90]. HPP was used to inactivate the histamineforming bacteria in tuna meat slurry, and HPP treatment resulted in morphological changes in the cells [91]. Direct HPP treatment caused morphological changes in the cell membrane, biochemical reactions, and the genetic mechanism of microorganisms, resulting in the microorganism inactivation. However, the effect is variable on microorganisms and depends upon different pressure and holding time. e total plate count and Enterobacteriaceae count of filleted tuna chunks decreased with an increase in pressure (100-300 MPa/5 min/25°C) compared to control tuna. e shelf life of filleted tuna chunks was increased up to 30 days at 2°C when packed in EVOH multilayered films after HPP treatment at 200 MPa [92]. However, in albacore tuna minced muscle, HPP treatments (275-310 MPa/2-6 min) resulted in a bacteriostatic effect on mesophiles and psychrophiles observed, i.e., were not able to kill microorganisms but were effective to prevent their proliferation during the shelf-life study. Treatment at 310 MPa for 6 min was most effective and improved the shelf life of minced albacore tuna for >22 days at 4°C and >93 days at −20°C. In addition, lipid stabilization, color, and texture improvement were also reported [93]. HPP can inactivate microorganisms and could be used for gelation without applying thermal treatment to achieve product characteristics close to fresh. HPP is currently employed in the USA by the oyster industry for shucking purposes, eliminating the need for costly skilled labor and reducing microbial risk to consumers by inactivating Vibrio spp. e oysters processed after HPP treatment in the USA (brand name plastic gold band) have received several national awards for quality products. Shelf-life of oysters was reported to be extended for 12 days at 4°C, and optimum conditions were found to be 300 MPa/2 min [94].
Based on the reported literature on meat, poultry, and seafood, it can be concluded that HPP is a very effective technique for providing microbial safety and under optimized conditions HPP leads to an extension in shelf life by almost double or more. e pressure is also very effective in denaturing the proteins responsible for holding the meat within the shell of oysters, mussels, crabs, and shrimp. erefore, a higher yield can be obtained as meat separation from the shell become more effortless and effective.
A study on the effect of HPP on the reduction of native microflora of Mexican multifloral honey was reported by [123]. is study evidenced that high-pressure processing at 600 MPa for 12 min resulted in a reduction of 0.8 log 10 of total mesophiles and 2.4 log 10 of Y&M counts. A similar study by Akhmazillah et al. [125] on manuka honey has also reported that a high-pressure of more than 350 MPa at 40°C for 3 min reduced the bacterial load from 6 logN CFU/g to 3 logN CFU/g. Pressure-mediated treatment (600 MPa for 5 min) extended the shelf life of ginger paste for six months under refrigerated conditions. Both HPP and pasteurization were equally effective in reducing microbial population, but retention of bioactive components was more in HPP [122]. Rocha-Pimienta et al. [124] worked on the inactivation of Bacillus cereus and S. aureus vegetative cells in human milk by HPP. e pressure intensity and holding times needed for maximum inactivation up to 5.81 and 6.93 log CFU/mL were 593.96 MPa for 3.88 min. Waite et al. [126] indicated that the HPP of ranch dressing reduced the Pediococcus acidilactici by more than 6.4 log CFU/g. e studies carried out by eminent scientists proved that HPP is a viable process to improve the food safety of food products with extended shelf life.
In baked goods, cakes and batters were studied for their microbial, physical, and structural changes due to HPP [127]. e mesophilic aerobic bacteria Y&M were more susceptible to high pressure, causing a reduction from 4.3 to 3.8 log CFU/g and 1.7 to 1.0 log CFU/g, respectively, at 600 MPa for 6 min. e wheat dough also showed a similar reduction in the total aerobic mesophilic count, and Y&M count within 1 min of treatment at all pressures studied (50-250 MPa) [128].
Cereals and pulses being nonperishable commodities were not extensively studied for microbial aspects of high pressure. However, HPP was studied for its effect on starch modification, improving nutritional quality, water absorption, gelatinization, and development of quick-cooking rice [129][130][131][132]. Ravichandran et al. [133] investigated the effect of high pressure on water absorption and gelatinization of Paddy (Basmati cv.). Presoaked and unsoaked grains were pressure treated at 350, 450, and 550 MPa for a temperature of 30, 40, and 50°C for a duration of 300, 600, 900, and 1200 s. e highest moisture content of up to 50% (dB) was achieved at 550 MPa/50°C/1200 s in addition to 25% of gelatinization. Yu et al. [134] studied the effect of high pressure on cooked rice dominated with Bacillus spp. with B. subtilis and B. cereus population. ey found that HPP treatment at 400 and 600 MPa increased the shelf life of cooked rice to 8 weeks at 25°C. HPP can be a very useful technique in reducing the microbial count of maize, honey, ranch dressing, and dough and in extending the shelf life of ginger paste and cooked rice.

Microbial Inactivation by HPP Assisted by Other Processing Techniques
HPP is an effective technique to inactivate or eliminate vegetative microorganisms but does not substantially affect spores [135]. pH in the case of fruits is low (<4.6) due to inherent acidity. It is further reduced by compression, so partially injured cells of microorganisms by HPP will not be able to recover in such a hostile environment. e difficulty is in the case of low acid products (poultry, meat, and milk) where pH values are >4.6. Spores grow even after HPP as soon as they find a suitable environment to grow and ultimately spoil the food [12]. To achieve higher efficiency for spore inactivation present in food samples by HPP alone, spores need to be germinated at low pressure in the first stage. en in the second stage, pressure needs to be elevated to inactivate germinated spores. But using HPP twice over a product increases the processing time and energy consumed and, subsequently cost of the product. Moreover, a combination of pressure and temperature (which can be increased along the HPP) eliminates the step of the spore's growth by HPP [136]. So, using a combination mode (HPP and temperature simultaneously) helps achieve rapid heating and cooling of products, reducing processing time and product cost [135].
Combining HPP with other nonthermal (irradiation, ultrasound, and pulsed electric field) and mild heat techniques (pasteurization, blanching, and drying) will be an additional hurdle for the microorganisms. Also, beyond 600 MPa pressure, there is an exponential increase in equipment cost, and not considered economical. Some authors proposed the use of antimicrobial preservatives (nisin, chitosans, and pediocin) to achieve a synergistic effect with pressure and to reduce process severity [13,[137][138][139][140]). Microbial inactivation by HPP assisted by other processing/ preservation techniques has been attempted by eminent researchers such as irradiation of chicken breast [141], irradiation of lamb meat [142], irradiation of kefir [14]; ultrasonication of Rhodotorula rubra [143,144]; use of preservatives such as lysozyme, ethylene diamine tetraacetic acid (EDTA) [145], and nisin [146]. Hauben et al. [145] found that cells were more sensitive toward pressurization in the presence of preservatives. Effectiveness of hurdle technology consisting of HPP (400 MPa/30 min/70°C), pH (4), and nisin (0.8I U/mL) to completely eradicate spore of Bacillus coagulans (2.5 CFU/mL), whereas pressure alone (400 MPa) at ambient temperature and neutral pH had no significant effect on viable spores [146]. Paul et al. [142] showed that either irradiation (1.0 kGy) or HPP (200 MPa for 30 min) only reduced staphylococci (10 4 /g) by 1 log cycle in lamb meat whereas, in combination, staphylococci can be completely eradicated. e complete deactivation of the microbial population (lactobacilli, lactococci, and yeast) of kefir was achieved using irradiation (5 kGy) and HPP (400 MPa/5 min/5°C) without changing structure and nutritional components (proteins and lipids) by Mainville et al. [14]. Treatment of Bacillus subtilis spores (400 MPa/30 min) and E. coli (300 MPa/10 min) using HPP followed by alternating current (50 Hz) leads to lethal damage to their cell component [147]. High pressure (500 kPa) in combination with heat (70°C) and ultrasound (117 db at 20 kHz) resulted in the inactivation of 99% of the Bacillus subtilis spore population [144]. Knorr [143] stated that HPP and ultrasonic individually were not adequate for inactivation of Rhodotorula rubra but complete inactivation was achieved in combination mode. e carbon dioxide-assisted HPP is one of the effective nonthermal technologies which has been applied successfully by different researchers for inactivating microorganisms and reported promising results [148]. is method utilizes moderate pressures (<50 MPa) sequentially or simultaneously with CO 2 to pasteurize liquid foods without compromising quality attributes. Pressure-ohmicthermal sterilization is a novel technology involving the utilization of high-pressure in combination or consecutive application of ohmic heating for low acid foods to achieve a sterilization effect and simultaneously reduce the severity of the individual effect of temperature on quality attributes [149]. A study on ultrafiltration in combination with HPP  Journal of Food Quality 11 with nisin compared to other treatments.

UHT milk Clostridium botulinum spores
To achieve 6 log 10 cycle reduction best optimum conditions were 545 MPa/51°C/ 13.3 min and nisin at 129 IU/ml concentration.

450-600/3-15
Beef steaks L. monocytogenes and enterohaemorrhagic E. coli It was suggested by the author to use allyl-isothiocyanate and carvacrol practically to assist with HPP to achieve extended shelf life. Li and Gänzle [197] 18 Journal of Food Quality was conducted by Zhao et al. [150] and reported apple juice to be microbiologically safe with better quality attributes than UF + HTST (high-temperature short time) juice throughout the storage period of 60 days. Evelyn and Silva [151] used HPP as a pretreatment to enhance thermosonication effectiveness to eliminate Alicyclobacillus acidoterrestris spores in orange juice. To inactivate spores of pathogenic bacteria (C. perfringens and B. cereus) and spoilage microorganisms, i.e., bacteria (Alicyclobacillus acidoterrestris), mold (Byssochlamys nivea and Neosartorya fischeri), and yeast (S. cerevisiae) present in food samples. HPP, thermal processing, high-pressure thermal processing, and thermal sonification was used. It was found that highpressure thermal processing (600 MPa/20 min/70-75°C) was more effective in achieving reductions. Moreover, a lower processing time was required to prepare a beef slurry, apple juice, strawberry puree, and beer [12]. Evelyn et al. [152] investigated the effect of high-pressure, high thermal treatments, and thermosonication treatments on the effect of B. nivea and N. fischeri mold spores. ey identified that spores age has a profound effect on inactivation through HPP. For B. nivea, the reduction was 2.7 log for 4week spores and 2 log for 12-week spores at 600 MPa/75°C/ 30 min. At the same treatment time, N. fischeri showed 2-4 log reduction, and 12-week-old spores were more resistant than 4-week-old spores indicating lower inactivation for older spores. On the other hand, thermosonic treatment at 0.33 W/ mL at 75°C was not effective in the inactivation of ascospores. e high pressure of 600 MPa and temperature of 75°C would be appropriate while targeting the most resistant spores, i.e., old spores of >12 weeks.
rough combination treatment requirement of high temperature was reduced as required in individual thermal processing to achieve the same degree of inactivation with better quality and less energy. Similar results were found in the literature for using antimicrobial agents and preservatives. Treatments like ultrasonication and modified atmosphere packaging in combination with HPP were also found to provide a significant positive result in spores inactivation compared to individual treatment over food. Some of the literature describing the use of different techniques along with HPP is given in Tables 4 and 5.

Benefits of Technology and Engineering Challenges
Uniform and instantaneous pressure transmission are effective in causing the death of pathogenic microorganisms due to the permeabilization of cell membranes without much increase in product temperature. HPP can even be carried out at low temperatures. Cell membrane permeability changes are reversible at low pressure but irreversible at high-pressure. e effect of pressure occurs only on noncovalent bonds, and covalent bonds are not affected. erefore, the characteristics of organoleptic and sensory properties remain unaltered, or the difference reported is not significant [16,198]. erefore, getting attention from the consumers and processors as the treated food is mildly processed and provides characteristics similar to fresh products. It is also effective in reducing enzyme activity, thereby enhancing the product's yield, quality, and shelf life, especially in fruits and vegetables [199]. Technology is environment friendly as no residues or waste are generated.
A variety of products can be treated using the technology, i.e., solid foods (preferably vacuum packaged) and liquid foods (in a flexible package, having the ability to bear compression up to 15 to 20%), dry-cured or cooked meat products, fish, seafood, marinated products, ready to eat meals, sauces, fruits and vegetables, juices, marmalades, jams, cheeses, milk, and other dairy products and nutraceutical [200,201]. Some foods that cannot be treated by high pressure are: food packaged in glass since glass containers will break on compression; products like bread and mousse that have air included in them; spices and dry fruits as these products have low moisture content. e equipment cost is high, and processed products have a niche market, so the product is commercially processed only in developed countries. It is due to the limited availability or development of large pressure vessels that can handle large volumes of food and withstand high pressures. Using one large pressure vessel rather than multiple small pressure vessels in parallel would be more effective and reduce operating and capital costs. e operating cost of the product is also dependent upon the operating parameters, i.e., amount of pressure, holding time, and temperature of the solvent used. erefore, it is pertinent to optimize processing variables [16]. Challenges to the commercial application of high-pressure technology include material handling, process optimization, limited knowledge in understanding kinetic data, the role of constituents cleaning, and disinfection of equipment.

Conclusions
is review illustrates the effectiveness of the nonthermal technique, i.e., HPP, on microorganism reduction and extension of the shelf life of different food products. Food composition, type and age of microorganism, amount of pressure, and treatment time play an important role in reducing the microorganism load. is novel technology is very effective against vegetative pathogens but has some limitations in the inactivation of spores. Effective and synergistic results in the inactivation of spores can be obtained when combined with other thermal and nonthermal techniques. is combination of hurdles reduces the severity of individual processing while retaining the nutritional quality of food products. Although initial equipment cost is high, recent advancements and an increase in the number of HPP units have resulted in the successful commercialization of HPP products in developed countries and are also getting acceptance worldwide. Still, further work can be done to reduce the equipment cost and further research on the resistance of microorganisms.

Data Availability
All data pertaining to this review are available within the article.
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