Isolation and structural determination of a novel flavonol triglycoside and 7 compounds from the leaves of oriental raisin tree (Hovenia dulcis) and their antioxidative activity

The hot water and ethanol extracts of oriental raisin tree (Hovenia dulcis Thunb) leaves showed DPPH radical scavenging activities. Antioxidants were purified and isolated from hot water and ethanol extracts by various column chromatographic procedures with the guided assay of DPPH radical scavenging. The structure of a novel flavonol triglycoside was determined to kaempferol 3-O-α-l-rhamnopyranoside-7-O-[α-d-glucopyranosyl(1→3)-α-l-rhamnopyranoside] (4). In addition, 7 known compounds were identified as caffeine (1), kaempferol 3,7-O-α-l-dirhamnopyranoside (2), kaempferol 3-O-α-l-rhamnopyranosyl( 1→6)-O-β-d-glucopyranosyl(1→2)-O-β-d-glucopyranoside (3), E-3-carboxy-2-petenedioate 5-methyl ester (5), quercetin 3-O-α-l-rhamnopyranoside (6), kaempferol 3-O-α-l-rhamnopyranoside (7), and quercetin 3-O-β-d-glucopyranoside (8). Compound 1–3 and 5–8 were newly identified in this plant. Quercetin glycosides (5, 7) showed higher DPPH radical scavenging activity than other compounds.     

A novel α-l-rhamnosidase with potential applications in citrus juice industry and in winemaking

The production of monoglycosylated flavonoids by α-l-rhamnosidases (EC 3.2.1.40) is an interesting development in biocatalysis. Applications of rhamnosidases in industry include removal of bitterness caused by naringin from citrus juices. In the present work, a psychrotolerant bacterial strain with α-l-rhamnosidase activity was isolated. The α-l-rhamnosidase was found to be able to degrade naringin and was purified and characterized. The α-l-rhamnosidase from Brevundimonas sp. Ci19 was able to release both rhamnose and prunin from naringin. The enzyme was partially purified with a performance of 2.7-fold purification. The α-l-rhamnosidase showed an optimum pH between 6.00 and 7.00 with substantial residual activity at pH 5.00 (85.3 %). The optimum temperature was between 20 and 37 °C. The enzyme showed activation in the presence of Ca²⁺ and Cd²⁺ ions and at a high ethanol concentration level (10 % v/v). Activity was found for β-d-glucosidase (EC 3.2.1.21) in the partially purified extract, but it was inactive in the acid pH region. This result indicates the potential for inactivation of β-d-glucosidase along with the high level of α-l-rhamnosidase activity necessary for the production of flavonoid glycosides. The α-l-rhamnosidase from Brevundimonas sp. Ci19 showed interesting properties for potential use not only in the citrus juice industry but also in winemaking.     

Microwave treatment of dietary gelatin does not generate cis-4-hydroxy-l-proline, an inhibitor of collagen biosynthesis

 Aqueous solutions (5 g/100 ml) of commercial preparations of (a) an enzymatic partial hydrolysate of gelatin and (b) type A gelatin were subjected to threefold heating to boiling in a domestic microwave oven at 750 W and to conventional heating. Then samples were totally hydrolyzed (6 M hydrochloric acid, 110  °C, 24 h) and investigated for the presence of eight possible stereoisomers of 3- and 4-hydroxyproline (Hyp) using capillary gas chromatography. Amino acids were analyzed as N(O)-trifluoroacetyl 2-propyl esters on Chirasil-l-Val and detected by selected ion monitoring mass spectrometry. Blanks of (a) and (b) were analyzed in parallel. Relative amounts of 5.0±0.2% cis-4-d-Hyp were generated from native trans-4-l-Hyp as a result of total hydrolysis in all samples and independent of previous treatment. Notably, neither cis-3-l-Hyp nor cis-4-l-Hyp could be detected in either of the gelatin samples. Thus a report on the generation of antifibrotic and therefore potentially hazardous cis-3-l-Hyp and cis-4-l-Hyp from protein-bonded native trans-3-l-Hyp and trans-4-l-Hyp on microwave heating of infant formulae could not be confirmed.     

Effect of l-lysine on the physicochemical properties of pork sausage

Effects of l-lysine on physicochemical properties of pork sausage were investigated. WHC values significantly increased using 0.8% l-lysine, but significantly decreased at 0.4% l-lysine (p<0.05), compared with controls. l-Lysine increased pH values and a* values, but significantly decreased CL, cohesiveness, L* values, and b* values (p<0.05), compared with controls. Addition of 0.4% l-lysine significantly (p<0.05) increased hardness, springiness, and chewiness values, compared with controls. SEM analysis showed that addition of 0.8% l-lysine induced formation of a smoother, more compact, and more uniform gel matrix, compared with controls. DSC analysis indicated that addition of 0.8% l-lysine significantly (p<0.05) increased thermal transition temperatures and enthalphy values, compared with controls, showing that interactions between l-lysine and myofibrillar proteins affected the properties of pork sausages.     

Enhanced production of l-tryptophan with glucose feeding and surfactant addition and related metabolic flux redistribution in the recombinant Escherichia coli

The production of l-tryptophan was investigated in a recombinant strain Escherichia coli W3110-ZDrr. It was observed that phosphate and feeding strategies are key factors to ensure the good cell growth and high production of l-tryptophan. The simple exponential feeding strategy could only produce 10.6 g/L l-tryptophan due to the improper feeding rate; while the manual glucose-feedback feeding approach could effectively control the substrate and inhibit the formation of acetate, and thus improvemed the l-tryptophan production to 25.5 g/L. The modified exponential feeding approach avoided overfeeding or underfeeding and achieved high production of l-tryptophan. Moreover, the addition of Tween 60 or PL61 could enhance the cell growth and the production of l-tryptophan in the fed-batch mode. Subsequent metabolic flux analysis showed that more carbon flux was distributed into the biosynthesis of l-tryptophan when Tween 60 or PL61 was supplied. The present work presents one base for further large-scale production of this important amino acid.     

Influence of buffer and l-phenylalanine concentration in the Rhizomucor miehei lipase catalysed synthesis of l-phenylalanyl-d-glucose ester investigated through response surface methodology

Reaction conditions for Rhizomucor miehei lipase (RML) catalysed synthesis of l-phenylalanyl-d-glucose using unprotected l-phenylalanine and d-glucose were optimized using response surface methodology (RSM). A central composite rotatable design (CCRD) was employed involving 32 experiments of five variables (l-phenylalanine concentration in mmol, amount of RML in mg, pH, incubation period in h and buffer concentration in mM) at five levels. A second-order polynomial equation was developed in terms of linear, quadratic and cross product terms to study the effects of variables on esterification yields. A R ² value of 0.7 was obtained for this complex reaction. From the surface and contour plots it was found that higher yields were observed for a very narrow pH range of 4.5–6.5 at l-phenylalanine concentrations above 3 mmol. The extent of esterification decreased with increase in RML concentration for incubation periods below 60 h. However, longer incubation periods above 60 h, enhanced esterification at all RML concentrations. Lower conversions below 0.5 mmol required less than 3.5 mmol l-phenylalanine concentration and a broad range of buffer concentration from 0.1 mM (0.1 ml, 0.1 M buffer of pH 6.0) to 0.5 mM (0.5 ml, 0.1 M buffer of pH 6.0). However, higher conversions from 0.6 to 0.8 mmol required a buffer concentration above 0.25 mM (0.25 ml, 0.1 M buffer of pH 6.0) at l-phenylalanine concentrations above 3.5 mmol. An optimum predicted yield of 1.01 mmol for l-phenylalanyl-d-glucose at 3 mmol l-phenylalanine, 100 mg of RML, 24 h incubation period, 0.5 mM (0.5 ml of 0.1 M buffer), pH 4.8 acetate buffer was found to agree with 0.97 mmol obtained under these experimental conditions. Validation experiments carried out under random conditions also exhibited good correspondence between predicted and experimental yields.     

Coumaroyl quinic acid derivatives and flavonoids from immature pear (Pyrus pyrifolia nakai) fruit

Fourteen compounds were isolated from 60% ethanol extracts of immature pear (Pyrus pyrifolia Nakai cv. Chuhwangbae) fruit using Amberlite XAD-2 column HPLC with guided DPPH radical scavenging assay. Based on MS and NMR analysis, the isolated compounds were identified as 5-O-trans-caffeoyl quinic acid methyl ester (1), malaxinic acid (2), 5-O-trans-p-coumaroyl quinic acid methyl ester (3), 5-O-cis-p-coumaroyl quinic acid methyl ester (4), 5-O-trans-p-coumaroyl quinic acid (5), trans-p-coumaric acid (6), methyl cis-p-coumarate (7), methyl trans-p-coumarate (8), 3,5-O-dicaffeoyl quinic acid (9), (-)-epicatechin (10), (S)-(+)-2-cis-abscisic acid (11), isorhamnetin 3-O-β-d-galacto-pyranoside (12), isorhamnetin-3-O-β-d-glucopyranoside (13), and isorhamnetin 3-O-α-l-rhamnopyranosyl (1→6)-O-β-d-glucopyranoside (14). Six compounds (1, 2, 6, 9, 10, and 13) were identified previously, but other compounds (3–5, 7, 8, 11, 12, and 14) were isolated for the first time from pear.     

Influence of various factors on formation of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) in a solid-state model system of Maillard reaction

The influences of various factors on the formation of 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) were investigated in a solid-state Maillard reaction system. Thermogravimetric analysis (TG) and differential scanning calorimetry analysis (DSC) showed that the solid-state Maillard reaction mainly occurred at around 150 °C. The DDMP formation from reducing sugars (glucose or fructose) and several selected amino acids (l-proline, l-alanine, l-asparagine, l-threonine, l-tyrosine and l-lysine) was compared. Proline was found to have special activity in DDMP formation when reacted with glucose because of its special catalytic action. The influences of reactant ratios, oxygen, reaction temperature and reaction time on DDMP formation from the reaction of proline with glucose were studied. Reactant molar ratios of glucose to proline played important impact on DDMP formation, and the formation of DDMP was improved when the ratio was higher than 1:1 and achieved the highest level at 2:1. Oxygen had no obvious effects on DDMP formation. The effects of reaction time and temperature were investigated together and revealed the information on the highest yield and minimum temperature for DDMP formation. Furthermore, the kinetics of DDMP formation was studied, and DDMP formation in the initial stage was shown to follow an apparent first-order reaction with an activation energy of 68.8 kJ/mol.     

Reduction of Acrylamide Formation in Sweet Bread with l-Asparaginase Treatment

Acrylamide, 2-propenamide, has the chemical formula $ \mathrm{CH}2=\mathrm{CH}-\mathrm{CO}-\mathrm{NH}2 $ . It is produced at elevated levels in high temperature fried and baked foods. It has adverse effects on human health and is proven to be neurotoxic, genotoxic, carcinogenic, and toxic to reproductive system. The aim of this paper was to reduce acrylamide formation in bakery products such as sweet bread by enzyme treatment. l-Asparaginase produced from Cladosporium sp. was treated to wheat-based dough at different concentrations (50–300 U). There was no change in the rheological properties of wheat flour and physico-sensory characteristics of bread with l-asparaginase treatment. Moisture, sugars, l-asparagine, acrylamide, and some indicators of Millard reaction (hydroxymethylfurfural (HMF), color, browning) were estimated. With increase in l-asparaginase level the acrylamide formation was reduced. At 300 U, there was 97 % and 73 % reduction of acrylamide formation in the crust and crumb regions of bread, respectively. HMF, a common intermediate product in the Maillard reaction and a genotoxic compound via 5-sulfoxymethylfurfural, also decreased in l-asparaginase-treated bread samples. These results indicated the potential of l-asparaginase enzyme for industrial and domestic applications in reducing harmful Maillard reaction compounds.     

Structure elucidation and immunological activity of a novel pectic polysaccharide from the stems of Avicennia marina

The structure of HAM-3-IIb-II, an acidic pectic polysaccharide in Avicennia marina, was characterized in detail and the immunological activity was investigated in this study. The results showed that HAM-3-IIb-II contained Rha, Ara, Gal, Glc, and GalA in a molar ratio of 1:2.8:2.4:6.1 and that HAM-3-IIb-II is a type I rhamnogalacturonan with branches of arabinose chains, galactosyl chains, and arabinogalactosyl chains. The backbone of HAM-3-IIb-II is mainly composed of disaccharide repeat units, →4)-α-d-GalA-(1 → 2)-α-l-Rhap-(1→. These repeats are predominantly linked to the O-4 of 1,2,4-linked α-l-Rhap in the branches of arabinose chains, galactosyl chains, and arabinogalactosyl chains. HAM-3-IIb-II had a significant effect on lipopolysaccharide-induced B lymphocyte proliferation, but it had little effect on concanavalin A-induced T lymphocyte proliferation compared with the control. This study provides a foundation for the exploration and utilization of A. marina in the future.     

Prebiotic effects of structurally identified galacto-oligosaccharides produced by β-galactosidase from Aspergillus oryzae

Novel galacto-oligosaccharides were produced by β-galactosidase from Aspergillus oryzae using lactose, and their structural characteristics and prebiotic effects were examined. Highly purified oligosaccharide fraction (HP) was prepared from a crude one (low purified, LP) by gel-filtration on Biogel P-2 column, which was further purified into S1 and S2 fractions by prep-HPLC. S1 and S2 were comprised of galactose (Gal) and glucose (Glc) in the ratio of 2 to 1. ESI-MS-MS and methylation analysis indicated that S1 and S2 were trisaccharides with structures of β-d-Galp-(1,6)-β-d-Galp-(1,4)-β-d-Glcp and β-d-Galp-(1,3)-β-d-Galp-(1,4)-β-d-Glcp, respectively. Herein, LP and HP were used as the carbon sources for determining the prebiotic activity score of probiotics including Lactobacillus and Bifidobacterium species. LP and HP at 1% and 2% (w/v) were observed at all positive scores on several probiotics, especially, B. infantis ATCC 15697 at the 2% level of HP (p<0.05). Consequently, structurally identified trisaccharides of HP can significantly enhance the growth of B. infantis.     

Development of an l-rhamnose bioluminescent microbial biosensor for analysis of food ingredients

A bioluminescent microbial biosensor induced specifically by l-rhamnose was developed and optimized, to monitor food processes where pectin is involved. The biosensor was developed by fusing the Escherichia coli promoter P_rhaB to the promoterless luxCDABE genes of Vibrio fischeri within plasmid pUCD615. Escherichia coli carrying this construct responded positively to l-rhamnose, but, because of low reporter signal, the enhancement of bioluminescence levels was required. The response was improved by promoter region length manipulation and resulted in 86-fold increase in response ratio. The specificity of the biosensor (80 times higher ratio response to l-rhamnose compared to other carbohydrates) and quantitative response to the target analyte were confirmed. The potential of this biosensor to monitor the hydrolysis of various types of pectin was demonstrated and a calibration curve was acquired. Our study presents a novel l-rhamnose-inducible microbial biosensor as a fast and economical alternative to chemical methods of pectin analysis.     

Combination of proanthocyanidins extracted from lotus seedpod and l-cysteine ameliorates memory impairment induced by alcohol and scopolamine in mice

Proanthocyanidins extracted from lotus seedpod (LSPC) have been shown to ameliorate cognitive deficits and oxidative damage. However, there is no scientific evidence of combined LSPC and l-cysteine in the treatment for Alzheimer’s disease (AD) in animal models. The first aim of this study was to study whether LSPC and l-cysteine combination was more potent than LSPC on improving memory impairment in alcohol-induced mice. The results showed LSPC and l-cysteine combination was more effective than LSPC, as indicated by same behavioral performance in Y-maze, but significantly enhanced total antioxidant capacity and superoxide dismutase (SOD) activity. The second goal of this study was to investigate whether different combination ratio of LSPC to l-cysteine impacts the cognition-enhancing effect in scopolamine-induced memory-deficit mice. Alterations in oxidative stress parameters and cholinergic activity in brains were also determined as possible mechanisms. Both M-Dose and H-Dose of LSPC and l-cysteine combination significantly improved scopolamine-induced memory impairment in Y-maze and step-down avoidance test by improving brain glutathione peroxidase and SOD activities, inhibiting brain malondialdehyde and hippocampus nitric oxide levels, inducible nitric oxide synthase activity in hippocampus and serum, and brain acetylcholinesterase activity. These results indicate that chronic administration of LSPC and l-cysteine combination ameliorates memory impairment, which may be related to inhibition of oxidative stress and improvement of cholinergic activity. These findings suggest LSPC and l-cysteine combination may provide a viable therapy in the treatment for AD and other forms of cognitive impairment.     

Melanin biosynthesis inhibitory effects of calycosin-7-O-β-d-glucoside isolated from astragalus (Astragalus membranaceus)

To discover an active skin depigmentation agent, we isolated a melanin biosynthesis inhibitor from the methanol extract of astragalus (Astragalus membranaceus) using a bioactivity-guided fractionation and identified it as calycosin-7-O-β-d-glucoside via spectroscopic analysis. The active compound exhibited tyrosinase inhibitory activity with an IC₅₀ value of 68 μM. In addition, calycosin-7-O-β-d-glucoside showed a melanin biosynthesis inhibition zone in a culture plate of Streptomyces bikiniensis. Furthermore, 75.78 μM of calycosin-7-O-β-d-glucoside dramatically decreased 50% of the melanin content on Melan-a cells without any apparent cytotoxicity. Theses results indicate that the calycosin-7-O-β-d-glucoside isolated from astragalus may be a good candidate for skin-whitening agent.     

Phytoestrogenic activity of Aceriphyllum rossii and rapid identification of phytoestrogens by LC–NMR/MS and bioassay-guided isolation

Aceriphyllum rossii Engler (Saxifragaceae) has been used as a nutritious food in Korea, but only minimal studies on this plant have been undertaken. We have examined the estrogenic effect of A. rossii and have discovered its potent constituents. To identify the estrogenic activity of A. rossii, we measured the transactivation of the estrogen-responsive element (ERE) in MCF-7 cells transfected with an ERE-containing construct after treatment of the extract and fractions of A. rossii. A. rossii showed a potent estrogenic activity and gallic acid ethyl ester; quercetin and kaempferol were identified as major constituents of the active fractions by liquid chromatography–nuclear magnetic resonance spectroscopy/mass spectrometry. We confirmed quercetin and kaempferol are the major active constituents after activity-guided isolation of seven compounds: gallic acid ethyl ester (1), quercetin (2), kaempferol (3), quercetin-3-O-(6″-galloyl)-β-d-glucopyranoside (4), quercetin-3-O-β-d-glucopyranoside (5), kaempferol-3-O-(6″-galloyl)-β-d-glucopyranoside (6), and kaempferol-3-O-β-d-glucopyranoside (7). These results show A. rossii could be a possible candidate for the treatment of postmenopausal symptoms.     

Purification, chemical characterization, and antioxidant activity of a polysaccharide from the fruiting bodies of sanghuang mushroom (Phellinus baumii Pilát)

A purified water-soluble polysaccharide was isolated from the fruiting bodies of sanghuang mushroom (Phellinus baumii Pilát) using hot water extraction, DEAE Sepharose Fast Flow anion exchange and High-Resolution Sephacryl S-1000 gel-filtration chromatography. The purified polysaccharide was a complex β-d-glucan, with a molecular mass of 230 kDa. Fourier-transform infrared (FT-IR), methylation analysis, and NMR spectroscopy of sanghuang mushroom polysaccharide (PBF3) indicated that the polysaccharide contained (1→3)-β-d-, (1→4)-β-d-, and branched (1→3,6)-β-d-glucopyranosyl residues. On the basis of hydroxyl radical assay, superoxide radical assay, and DPPH radical assay, its antioxidant activities were investigated. PBF3 had significant effect on scavenging hydroxyl radicals, an equivalent inhibiting ability to vitamin C on superoxide radical, and a little lower scavenging activity on DPPH radical than vitamin C, and should be explored as a novel potential antioxidant.     

Two novel glycosyl cinnamic and benzoic acids from korean black raspberry (Rubus coreanus) wine

Korean black raspberry (Rubus coreanus) wine was solvent-fractionated with n-hexane, ethyl acetate, and n-butanol and the n-butanol layer was purified by various column chromatographic procedures, including Amberlite XAD-2, Sephadex LH-20, and octadecylsilane resins as well as high performance liquid chromatography. Two novel glycosyl cinnamic and benzoic acids were isolated and their structures were (E)-8-O-β-d-glucopyranosylcinnamic acid (1) and 3′-[O-β-d-glucopyranosyl)(1″→6′)-α-d-psicofuranosyl] benzoate (2) based on the spectroscopic data obtained by high resolution fast-atom bombardment mass spectroscopy and nuclear magnetic resonance analyses.     

l(+)-Lactic acid production from furfural residues and corn kernels with treated yeast as nutrients

In this study, Streptococcus thermophilus and Lactobacillus bulgaricus were used to produce l(+)-lactic acid by simultaneous saccharification and fermentation (SSF). The use of hydrolyzed yeast as cheap nutrients and mixtures of cellulosic materials and starchy materials as carbon source for l(+)-LA production was evaluated. Heat treatment (121 °C) was proven to be an effective method to improve the performances of yeast as nutrients for the fermentations using different carbon sources. The addition of yeast hydrolyzate obviously lowered the surface tension of medium and improved enzyme hydrolysis of furfural residue (FR) as the concentration was beyond 10 g/L. Carbon–nitrogen ratio, substrates composition, substrates feeding rate and enzyme-feeding strategy will affect the productivity of l(+)-LA production from mixed substrates. SSF of FR and corn saccharification liquid tends to obtain good yields, when the total WIS content is in 10 % and carbon–nitrogen ratio is about 30. This study provides an encouraging means of producing l(+)-LA from lignocellulosic resource and starchy resource, which could optimize the use of raw materials.     

Synergistic effect of lecithins for tocopherols: lecithin-based regeneration of α-tocopherol

Phospholipids synergistically enhance the antioxidant effect of phenolic antioxidants. Mixtures of lecithins, ascorbic acid and tocopherols are known as strong antioxidants. In our study, the mechanism of the synergistic effect of phospholipids with tocopherols was investigated. The oxidative deterioration and the degradation of tocopherols were analyzed in degummed rapeseed oil or in mixtures with ethyl linoleate at 25 or 110?°C. It was shown that lecithins reduce the degradation of tocopherols in rapeseed oil or in ethyl linoleate model system at 25 or 110?°C. It was found that phosphatidylethanolamine (PE) and phosphatidylserine (PS) can regenerate α-tocopheryl quinone (α-TQ) to α-tocopherol at elevated temperatures. The regeneration could be catalyzed by acetic acid. The synergistic effect of PE or PS with α-tocopherol and l-ascorbic acid resulted from the regeneration of α-TQ to α-tocopherol, because PE, l-ascorbic acid and α-tocopherol show the same stabilization effect upon ethyl linoleate as PE, l-ascorbic and α-TQ. Phosphatidylcholine as a non-amine phospholipid does not regenerate α-TQ. However, it improves the regeneration of α-tocopherol from α-tocopheroxyl radicals with l-ascorbic acid in ethyl linoleate through its ability to acts as a phase transfer catalyst between the polar l-ascorbic acid and the unpolar α-tocopheroxyl radicals. A mechanism for the regeneration of α-TQ by PE or PS is suggested. The activation of α-TQ by PE or PS and the formation of α-tocopherones are the key steps of the mechanism.     

Freezing Characteristics and Storage Stability of Broccoli (Brassica oleracea L. var. botrytis L.) Under Osmodehydrofreezing and Ultrasound-Assisted Osmodehydrofreezing Treatments

Freezing characteristics (freezing time, latent heat of fusion of ice, and freezable water content) and quality parameters (drip loss, color, firmness, and l-ascorbic acid content) of broccoli (osmodehydrofrozen and ultrasound-assisted osmodehydrofrozen) during frozen storage were investigated. Freezing time, latent heat of fusion of ice, and freezable water content of osmodehydrated samples decreased significantly compared to samples which were not under osmotic dehydration. The changes of drip loss, color, firmness, and l-ascorbic acid content of osmodehydrofrozen and ultrasound-assisted osmodehydrofrozen broccoli during frozen storage were inhibited markedly compared to samples which were not submitted to osmotic dehydration before freezing. Compared to osmotic dehydration, the ultrasound-assisted osmotic dehydration shortened the needed dehydration time and better preserved the firmness and l-ascorbic acid content after osmotic dehydration pretreatment. In addition, the ultrasound-assisted osmotic dehydration minimized the drip loss and loss of l-ascorbic acid content and better maintained the color and firmness when stored at ?25 °C for 6 months. These findings indicate that it is promising to apply ultrasound-assisted osmodehydrofreezing in freezing and frozen storage of food.