High Hydrostatic Pressure Treatment Research Articles

Applications of Lactobacillus acidophilus-Fermented Mango Protected Clostridioides difficile Infection and Developed as an Innovative Probiotic Jam.

Clostridioides difficile infection (CDI) is a large intestine disease caused by toxins produced by the spore-forming bacterium C. difficile, which belongs to Gram-positive bacillus. Using antibiotics treatment disturbances in the gut microbiota and toxins produced by C. difficile disrupt the intestinal barrier. Some evidence indicates fecal microbiota transplantation and probiotics may decrease the risk of CDI recurrence. This study aimed to evaluate the efficacy of fermented mango by using the lactic acid bacteria Lactobacillus acidophilus and develop innovative products in the form of fermented mango jam. L. acidophilus-fermented mango products inhibited the growth of C. difficile while promoting the growth of next-generation probiotic Faecalibacterium prausnitzii. Both supernatant and precipitate of mango-fermented products prevented cell death in gut enterocyte-like Caco-2 cells against C. difficile infection. Mango-fermented products also protected gut barrier function by elevating the expression of tight junction proteins. Moreover, L. acidophilus-fermented mango jam with high hydrostatic pressure treatment had favorable textural characteristics and sensory quality.

Application of high hydrostatic pressure for the reduction of STEC on raw ground beef patties and its impact on physicochemical properties: pH and color

This study on raw ground beef patties evaluated: resistance of STEC strains inoculated at 6 log CFU/g (O157:H7, O26, O45, O103, O111, O121, and O145) subjected to 350, 450 and 600 MPa, for 5 min at 10 °C; effectiveness of pressure for initial loads (2, 3 and 4 log CFU/g) of O157:H7; and general impact of high hydrostatic pressure (HHP) on raw patties. For 600 MPa treatments and 6 log CFU/g load of STEC, ~5 log CFU/g reductions were obtained with all strains. For 450 MPa, differences in baroresistance among strains became evident. When inoculating O157:H7 at different loads, counts after 600 MPa fell below quantification limits, though virulence genes were detected for the two highest loads. Additionally, HHP reduced native aerobic microbiota to <1 log CFU/g and slightly affected a* and b* color values. Montecarlo simulation was used to estimate potential initial counts of STEC that allow compliance with existing regulatory limits after applying HHP, showing that absence of STEC studied can be achieved in 65 g of patties provided initial loads are ~2 log CFU/g. Finally, HHP treatments at 600 MPa and mild temperatures can be considered a valid non-thermal processing technology to achieve 5 log CFU/g reductions.

High hydrostatic pressure combined with moisture regulators improves the tenderness and quality of beef jerky

The influence of high hydrostatic pressure (HHP) at different pressure levels (0.1, 100, 200, and 300 MPa) combined with moisture regulators (MR) on the tenderness, water content, and quality of beef jerky was investigated. HHP treatment reduced the shear force (SF) of beef jerky (P < 0.05). The beef jerky treated with MR+HHP exhibited higher tenderness than the beef jerky treated only with HHP (P < 0.05). The MR+HHP samples had significantly higher moisture content than the HHP samples (P > 0.05) when the water activity was maintained at approximately 0.7. MR+HHP contributed to a shorter T21 value and a higher P21 value, which indicated an improvement in the water-binding ability of the beef muscle. Analysis of the microstructure showed that MR+HHP led to the fracture of the Z-line and destruction of the sarcomere structure. Sensory analysis showed that MR+HHP-200 samples had significantly higher tenderness and overall acceptable scores than other samples (P < 0.05).

Changes induced by high hydrostatic pressure in acidified and non‐acidified milk during Oaxaca cheese production

SummaryOaxaca cheese, produced using the pasta filata method, is a very popular Mexican dairy products. In this work, the effect of high hydrostatic pressure (HHP) and the acidification process before and after HHP treatment of raw cow milk was studied at different pressure levels (150, 300 and 500 MPa) and holding times (10 and 30 min). Clotting time, proximal composition, microstructure, secondary protein structure and electrophoretic profile were evaluated. HHP did not influence clotting time in samples acidified before HHP, but it appears to have a positive effect at lower pressure treatments on non‐acidified milk. Moisture, protein and fat were similar in cheeses treated at most HHP conditions regardless of the acidification. HHP did not influence the microstructure of cheese and the secondary structure of proteins. The use of HHP during the manufacture of Oaxaca cheese allowed preserving quality parameters evaluated without advantages in processing time and the product's proximal composition.

High Hydrostatic Pressure Treatment Ensures the Microbiological Safety of Human Milk Including Bacillus cereus and Preservation of Bioactive Proteins Including Lipase and Immuno-Proteins: A Narrative Review.

Breast milk is the nutritional reference for the child and especially for the preterm infant. Breast milk is better than donated breast milk (DHM), but if breast milk is not available, DHM is distributed by the Human Milk Bank (HMB). Raw Human Milk is better than HMB milk, but it may contain dangerous germs, so it is usually milk pasteurized by a Holder treatment (62.5 °C 30 min). However, Holder does not destroy all germs, and in particular, in 7% to 14%, the spores of Bacillus cereus are found, and it also destroys the microbiota, lipase BSSL and immune proteins. Another technique, High-Temperature Short Time (HTST 72 °C, 5–15 s), has been tried, which is imperfect, does not destroy Bacillus cereus, but degrades the lipase and partially the immune proteins. Therefore, techniques that do not treat by temperature have been proposed. For more than 25 years, high hydrostatic pressure has been tried with pressures from 100 to 800 MPa. Pressures above 400 MPa can alter the immune proteins without destroying the Bacillus cereus. We propose a High Hydrostatic Pressure (HHP) with four pressure cycles ranging from 50–150 MPa to promote Bacillus cereus germination and a 350 MPa Pressure that destroys 106 Bacillus cereus and retains 80–100% of lipase, lysozyme, lactoferrin and 64% of IgAs. Other HHP techniques are being tested. We propose a literature review of these techniques.

High hydrostatic pressure shapes the development and production of secondary metabolites of Mariana Trench sediment fungi

The hadal biosphere is one of the least understood ecosystems on our planet. Recent studies have revealed diverse and active communities of prokaryotes in hadal sediment. However, there have been few studies on fungi in hadal sediment. Here we report the first isolation and cultivation of 8 fungi from the Mariana Trench sediment. The individual colonies were isolated and identified as Stemphylium sp., Cladosporium sp., Arthrinium sp., Fusarium sp., Alternaria sp., and Aspergillus sp. High hydrostatic pressure (HHP) test was carried out to identify the piezophily of these hadal fungi. Among them, 7 out of the 8 fungal isolates exhibited the ability of germination after incubation under 40 MPa for 7 days. Vegetative growth of the isolates was also affected by HHP. Characterization of secondary metabolites under different pressure conditions was also performed. The production of secondary metabolites was affected by the HHP treatment, improving the potential of discovering novel natural products from hadal fungi. The antibacterial assay revealed the potential of discovering novel natural products. Our results suggest that fungal growth pressure plays an important role in the development and production of secondary metabolites of these hadal fungi under the extreme environment in the Mariana Trench.

High‐pressure processing of shalgam with respect to quality characteristics, microbial inactivation, and shelflife extension

High hydrostatic pressure (HHP) processing of traditional fermented turnip juice (shalgam) was studied with respect to quality attributes, microbial inactivation, and shelflife extension. According to Box–Behnken design, shalgam samples were processed using 3–15 min, 4–40°C, and 200–500 MPa. The optimum processing conditions involved 34.23°C, 15 min, and 500 MPa for the color intensity and tone of 3.43 and 0.69 and the numbers of viable total mold and yeast, Lactococcus lactis subsp. cremoris, and Lactobacillus paracasei of 3.09, 2.51, and 2.68 log cfu/ml, respectively. Shalgam samples processed under the optimum conditions were stored at 4 and 22°C for 90 days. The control samples at 4 and 22°C were spoiled at 60 and 45th days, respectively. The storage time rather than the HHP processing affected physical properties, organic acids, phenolic compounds, and anthocyanins of shalgam. The treatment samples had significantly lower microbial growth during the storage time. Practical applications Due to the deteriorated color, bioactive, and sensory properties, the traditional heat processing of shalgam with the addition of antimicrobial agents (such as sodium benzoate) to extend its shelflife is not desirable by consumers. Alternative processing technologies are needed to extend its shelflife with no significant changes in its important properties. Its HHP treatment appeared as a practical option that preserved its quality parameters and extended its shelflife.

High hydrostatic pressure treatment of Arthrospira (Spirulina) platensis extracts and the baroprotective effect of sugars on phycobiliproteins

The effect of High Hydrostatic Pressure (HHP) (600 MPa; 300 s) upon phycobiliproteins from Spirulina platensis extract, in aqueous systems, has been investigated along with the potential baroprotective ability of several sugars (sucrose, trehalose and glucose) at different concentrations (20%, 40% w/w). Phycobiliproteins stability and technological functionality were assessed through spectroscopical and colour analysis, and radical scavenging ability.HHP treatment resulted in a decrease of the phycobiliproteins concentration with allophycocyanin being the most sensitive towards HHP stress (p ≤ 0.05). Addition of saccharides had a positive effect on phycocyanin stability with a higher residual concentration after HHP treatment (p ≤ 0.05) irrespective of the type and concentration of sugars added in the system, whilst baroprotective effects on allophycocyanin and phycoerythrin were observed only at the highest (40%) concentrations tested (p ≤ 0.05). After the HHP treatment, colouring ability of Spirulina platensis extract resulted in being directly correlated to phycocyanin concentration variation (R2 = 0.75). Addition of sugars preserved the antioxidant activity of the extract subject to high pressure regardless of the sugar type. Industrial relevanceSpirulina platensis has become a popular ingredient among manufacturers and consumers, especially for its technological functionality and nutritional properties. Nevertheless, processing and storage may result detrimental for its performance in foods. High hydrostatic pressure is a successful, non-thermal technology that is finding increasing applications in the food industry though processing costs of food products stabilized by HHP result, nowadays, being 7 to 15-fold higher than thermal pasteurization. However, consumers today are willing to pay higher prices for higher quality products with better sensory profiles and nutritional properties. Moreover, compared to thermal pasteurization, HHP is a rather new technology whose cost-effectiveness and environmental impact is expected to improve over time, with a consequent reduction of the gap among performance of these two technologies. Hence, this study focused on the effects of HHP treatments on aqueous Spirulina platensis extracts, alone or added with small saccharides to evaluate their potential baroprotective abilities, providing results of interest for application in food product design and development.

Induced Changes in Aroma Compounds of Foods Treated with High Hydrostatic Pressure: A Review.

Since conventional thermal processing can have detrimental consequences on aroma compounds, non-thermal technologies such as high hydrostatic pressure (HHP) have been explored. HHP may alter the weak chemical bonds of enzymes. These changes can modify the secondary, tertiary, and quaternary structures of key enzymes in the production of aroma compounds. This can result in either an increase or decrease in their content, along with reactions or physical processes associated with a reduction of molecular volume. This article provides a comprehensive review of HHP treatment’s effects on the content of lipid-derived aroma compounds, aldehydes, alcohols, ketones, esters, lactones, terpenes, and phenols, on various food matrices of vegetable and animal origin. The content of aldehydes and ketones in food samples increased when subjected to HHP, while the content of alcohols and phenols decreased, probably due to oxidative processes. Both ester and lactone concentrations appeared to decline due to hydrolysis reactions. There is no clear tendency regarding terpenes concentration when subjected to HHP treatments. Because of the various effects of HHP on aroma compounds, an area of opportunity arises to carry out future studies that allow optimizing and controlling the effect.

Effect of HHP, Enzymes and Gelatin on Physicochemical Factors of Gels Made by Using Protein Isolated from Common Cricket (Acheta domesticus).

The effect of high hydrostatic pressure (HHP) combined with enzymatic methods or gelatin incorporation in the gelation process of protein isolated from Acheta domesticus was investigated. The results indicate that transglutaminase (TGasa) or glucose oxidase (GOx) enzymes can induce reversible aggregation in dispersions of insoluble protein fractions and increase viscosity in dispersions of soluble fractions, but does not induce gel formation even after HHP treatment; in consequence, enzymatic treatment on cricket protein can be used to increase viscosity but not to form gels. It is technically feasible to obtain gels by adding 2% porcine gelatin to dispersions of protein fractions and subjecting them to HHP. The firmness and syneresis variation values of those gels during storage depended on the protein extracted fraction (insoluble or soluble protein) and on the concentration of protein used. The highest hardness and lowest syneresis was found with the gels obtained from the insoluble fraction at 11 and 15% (w/w) protein concentration. Color difference (ΔE* > 3) appreciable to the naked eye was observed along the storage period and no noticeable pH variations were found after 28 days of storage. Results indicate that new raw materials of interest can be developed for the food industry based on cricket protein isolates, to make high-protein foods which could be applied in a wide variety of different food applications including 3D printing or fat substitution.

Bacterial Spore Inactivation in Orange Juice and Orange Peel by Ultraviolet-C Light.

Spore-forming bacteria are a great concern for fruit juice processors as they can resist the thermal pasteurization and the high hydrostatic pressure treatments that fruit juices receive during their processing, thus reducing their microbiological quality and safety. In this context, our objective was to evaluate the efficacy of Ultraviolet-C (UV-C) light at 254 nm on reducing bacterial spores of Alicyclobacillus acidoterrestris, Bacillus coagulans and Bacillus cereus at two stages of orange juice production. To simulate fruit disinfection before processing, the orange peel was artificially inoculated with each of the bacterial spores and submitted to UV-C light (97.8–100.1 W/m2) with treatment times between 3 s and 10 min. The obtained product, the orange juice, was also tested by exposing the artificially inoculated juice to UV-C light (100.9–107.9 W/m2) between 5 and 60 min. A three-minute treatment (18.0 kJ/m2) reduced spore numbers on orange peel around 2 log units, while more than 45 min (278.8 kJ/m2) were needed to achieve the same reduction in orange juice for all evaluated bacterial spores. As raw fruits are the main source of bacterial spores in fruit juices, reducing bacterial spores on fruit peels could help fruit juice processors to enhance the microbiological quality and safety of fruit juices.

Effects of high hydrostatic pressure on microstructure, physicochemical properties and in vitro digestibility of oat starch/β‐glucan mixtures

AbstractOats do not contain gluten protein, and oat dough structure is formed mainly through the hydrogen bonding of starch and β‐glucan. As a non‐thermal processing technology, high hydrostatic pressure (HHP) is mainly used to modify starch and protein in food processing. This study investigated the effects of HHP treatment on the morphological, structural, thermal, pasting and in vitro digestion properties of oat starch/β‐glucan mixtures. Results showed that β‐glucan interconnects with amylose through hydrogen bonding and has a protective effect on the crystalline region of oat starch. Effect of HHP treatment on the crystal structure of mixture system goes through crystal structure perfection stage, crystallisation disintegration and gelatinisation stage. After 300–400 MPa treatment, the changes in particle surface were not obvious, the phase transition temperature, the ΔHgel and the PT of mixtures increased, while the particle size, viscosity and BD values decreased. After 500–600 MPa treatment, mixtures were completely gelatinised, most of the particles swelled and deformed, the particle size increased significantly. The principal component analysis results show that the complexes were distributed in the same region with similar properties after the 300–400 MPa and 500–600 MPa treatments, respectively.

Inactivation of Bacillus cereus spores using a combined treatment of UV-TiO2 photocatalysis and high hydrostatic pressure

Bacillus cereus spores are resistant to high hydrostatic pressure (HHP) processing treatment. A combination of UV-TiO2 photocatalysis (UVTP for 10 min) and two cycles of 600 MPa HHP treatment for 10 min for the first cycle and 1 min for the second cycle (UVTP-2HHP) at ambient temperature was applied to inactivate B. cereus spores inoculated on a solidified agar matrix (SAM) used as a model matrix. Two cycles of HHP treatment were used as a strategy for induction of spore germination, followed by inactivation. UVTP and 2 cycles of HHP resulted in a 5.0-log CFU/cm2 spore reduction (initial spore count was 6.6 log CFU/cm2), including an approximate 0.8-log CFU/cm2 reduction due to a synergistic effect. The inactivation mechanism of UVTP pretreatment was related to lipid peroxidation of the spore membrane based on the level of malondialdehyde (MDA) making spores susceptible to the HHP treatment. Flow cytometry and transmission electron microscopic (TEM) analyses showed severe physiological alteration and structural damage to spores after the combined treatment. UVTP and 2 cycles of HHP showed potential for effective inactivation of B. cereus to ensure food safety from B. cereus spores on food products. Practical applicationsInactivation of bacterial spores remains a technical challenge for HHP and other interventions because spores are highly resistant to high pressure. Pretreatment with UVTP followed by two cycles of HHP resulted in reduction in B. cereus spores due to a synergistic effect. This hurdle technology of UVTP and HHP can help food industry in ensuring food safety against the Bacillus spores.

Effects of high hydrostatic pressure treatment on the emulsifying behavior of myosin and its underlying mechanism

Myosin, a protein originating from muscle, is traditionally used for meat products for its ability to generate emulsions. Small changes in the emulsifying and conformational characteristics of proteins greatly influence the gel-emulsifying meat products. The effect of high hydrostatic pressure (HHP) on the emulsifying behavior of myosin prepared at different pressures (0.1, 100, 150, and 200 MPa) was investigated. The particle size, microstructure, interfacial tension, emulsifying properties, and secondary structure of myosin were analyzed. The results showed that as pressure increased from 0.1 to 150 MPa, the size of oil drops was reduced, the viscoelasticity of interfacial protein film improved, and the emulsifying activity and stability of myosin increased. Meanwhile, as the pressure increased from 0.1 to 150 MPa, the α helix content of the protein decreased significantly (P < 0.05), which was beneficial for the intermolecular interaction between proteins. A further increase in pressure (to 200 MPa) was detrimental to emulsifying activity and stability. As the pressure increased to 200 MPa, the emulsion displayed a decreased interfacial elasticity and increased protein random coil contents, and the particle size noticeably increased (P < 0.05). Therefore, it is concluded that HHP treatment at 150 MPa improves the stability of myosin emulsions.

Optimization of High Hydrostatic Pressure Treatments on Soybean Protein Isolate to Improve Its Functionality and Evaluation of Its Application in Yogurt.

This work aimed to improve the functional properties of soybean protein isolate (SPI) by high hydrostatic pressure (HHP) and develop SPI incorporated yogurt. Response surface methodology (RSM) was used to optimize the HHP treatment parameters, including pressure, holding time, and the ratio of SPI/water. Water holding capacity, emulsifying activity index, solubility, and hardness of SPI gels were evaluated as response variables. The optimized HPP treatment conditions were 281 MPa of pressure, 18.92 min of holding time, and 1:8.33 of SPI/water ratio. Water and oil holding capacity, emulsifying activity, and stability of SPI at different pH were improved. Additionally, relative lipoxygenase (LOX) activity of HHP treated SPI (HHP-SPI) was decreased 67.55 ± 5.73%, but sulphydryl group content of HHP-SPI was increased 12.77%, respectively. When incorporating 8% of SPI and HHP-SPI into yogurt, the water holding capacity and rheological properties of yogurt were improved in comparison with yogurt made of milk powders. Moreover, HHP-SPI incorporated yogurt appeared better color and flavor.

Effect of high hydrostatic pressure (HHP) and supercooling storage in leaf mustard (Brassica juncea L.) kimchi: Modelling of microbial activity and preservation of physicochemical properties

Kimchi products have a short shelf life due to continuous bacterial growth and fermentation. Herein, the microbiological and physicochemical properties of leaf mustard (Brassica juncea L.) kimchi were evaluated after high hydrostatic pressure (HHP) treatment (400 and 600 MPa for 5 min) and storage under supercooling (˗4.5 °C) or refrigeration (4 and 8 °C) conditions. Moreover, predictive models describing the growth kinetics of total lactic acid bacteria (TLAB) in leaf mustard kimchi were constructed. Treatment with 600 MPa reduced the total LAB and coliform counts in the samples before storage to 1.30 and > 1 log CFU/g, respectively. Supercooling storage prevented microbial growth for 30 days and allowed the maintenance of the titratable acidity, reducing sugar content, instrumental colour, total phenolic content, and antioxidant capacity of the samples. The Logistic and Gompertz models were relatively more acceptable to predict the growth behaviour of TLAB in leaf mustard kimchi treated/non-treated with 400 MPa during supercooled or refrigerated storage. Altogether, these findings suggest that HHP treatment combined with supercooling storage conditions that delay O2 and CO2 concentration changes in packaged kimchi's headspace could help maintain the initial microbiological and physicochemical qualities of leaf mustard kimchi.

Monitoring, by high‐performance liquid chromatography, near‐infrared spectroscopy, and color measurement, of phytonutrients in tomato juice subjected to thermal processing and high hydrostatic pressure

Tomatoes are consumed as fresh and processed products, which contain nutritionally important phytonutrients. It is necessary to use a rapid and reliable analytical method to monitor the quality of tomato products. The study was conducted to study the feasibility of near-infrared spectroscopy (NIRS) and color measurement with data obtained from high-performance liquid chromatography (HPLC), for monitoring the change in tomato juice, as a consequence of thermal and high hydrostatic pressure (HHP) treatments at different conditions. Partial least squares regression was applied to assess the correlation between HPLC values of ascorbic acid (A.A.), lycopene (Lyc.), β-carotene (β-car.), and NIRS. The correlation was confirmed with R2P of .82, .92, and .91 based on the lowest values of the standard error of prediction (SEP) for A.A., Lyc., and β-car., respectively. The lowest degradation of A.A. (35% and 49%), Lyc. (12% and 3.6%), and β-car. (23.3% and 18.4%) was recorded for juice thermally processed and HHP treated, respectively. Novelty impact statement Overdosage of food processing methods and exposure times generate food products that are poor in nutritional value. The results confirmed the ability of the near-infrared spectroscopy to determine, measure, and predict the internal quality characteristics (ascorbic acid, lycopene, and β-carotene) values of processed tomatoes and choose the best treatment methods and doses that afford the best quality to the final product. With increasing consumer awareness and market needs for healthy, and high nutritional value foods, the food industry will have profited from the nondestructive methods for quality detection, which in turn will determine the best processing methods, the optimum dosages, and the appropriate exposure time for processing. Hence, this technique is beneficial to produce high nutritional value food products able to meet the market needs, competition, and consumer satisfaction.

Different sequence of high-hydrostatic pressure and mild-heat treatment on the colour and sensory characteristics of strawberry puree

AbstractThe effect of high pressure processing (300 and 600 MPa) combined with mild heat treatment (55 and 75 °C) on the colour parameters, anthocyanin content, and sensory characteristics of strawberry puree were examined after the treatments and 2 weeks of cold storage at 2 and 15 °C. As on an industrial scale the simultaneous implementation of these treatments remains a challenge, the HHP and heat treatments were carried out consecutively in different sequences. The colour parameters and the anthocyanin content did not change significantly due to the treatments, but decreased during cold storage, at 15 °C storage the changes were more intensive than at 2 °C. Regarding the sensory test results, the different sequence of the 600 MPa-75 °C combined treatments is not detectable even after 2 weeks of storage at 15 °C, but in case of 300 MPa-55 °C, the enzyme inactivation is probably not sufficient enough as differences between the samples were detected.

Mechanical Characterization of Human Trabecular and Formed Granulate Bone Cylinders Processed by High Hydrostatic Pressure.

One main disadvantage of commercially available allogenic bone substitute materials is the altered mechanical behavior due to applied material processing, including sterilization methods like thermal processing or gamma irradiation. The use of high hydrostatic pressure (HHP) might be a gentle alternative to avoid mechanical alteration. Therefore, we compressed ground trabecular human bone to granules and, afterwards, treated them with 250 and 300 MPa for 20 and 30 min respectively. We characterized the formed bone granule cylinders (BGC) with respect to their biomechanical properties by evaluating stiffness and stress at 15% strain. Furthermore, the stiffness and yield strength of HHP-treated and native human trabecular bone cylinders (TBC) as control were evaluated. The mechanical properties of native vs. HHP-treated TBCs as well as HHP-treated vs. untreated BGCs did not differ, independent of the applied HHP magnitude and duration. Our study suggests HHP treatment as a suitable alternative to current processing techniques for allogenic bone substitutes since no negative effects on mechanical properties occurred.

Inactivated Nevus Tissue with High Hydrostatic Pressure Treatment Used as a Dermal Substitute after a 28-Day Cryopreservation Period.

Background Giant congenital melanocytic nevi (GCMN) treatment remains controversial. While surgical resection is the best option for complete removal, skin shortage to reconstruct the skin defect remains an issue. We report a novel treatment using a high hydrostatic pressurization (HHP) technique and a cryopreservation procedure. However, cryopreservation may inhibit revascularization of implanted nevus tissue and cultured epidermal autograft (CEA) take. We aimed to investigate the influence of the cryopreservation procedure on the HHP-treated dermis specimen and CEA take on cryopreserved tissue. Methods Nevus tissue harvested from a patient with GCMN was inactivated with HHP of 200 MPa and then cryopreserved at -30°C for 28 days. The cryopreserved specimen was compared with fresh (HHP-treated without cryopreservation) tissue and with untreated (without HHP treatment) tissue to evaluate the extracellular matrix, basal membranes, and capillaries. Cultured epidermis (CE) take on the cryopreserved tissue was evaluated following implantation of the cryopreserved nevus tissue with CE into the subcutis of nude mice. Results No difference was observed between cryopreserved and fresh tissue in terms of collagen or elastic fibers, dermal capillaries, or basement membranes at the epidermal-dermal junction. In 4 of 6 samples (67%), applied CE took on the nevus tissues and regenerated the epidermis in the cryopreserved group compared with 5 of 6 samples (83%) in the fresh group. Conclusion Cryopreservation at -30°C for 28 days did not result in significant damage to inactivated nevus tissue, and applied CE on the cryopreserved nevus tissues took and regenerated the epidermis. Inactivated nevus tissue with HHP can be used as a dermal substitute after 28-day cryopreservation.