Based on the hypothesis that cross-links contribute considerably to the physicochemical and functional properties of protein, we examined the usefulness of the cross-linking enzyme transglutaminase (TGase) in terms of protein polymerization, gelation, film formation, and peptide incorporation into protein. TGase-producing microorganisms were discovered, and mass production and preparation of the enzyme was accomplished. TGase has been used in the production of various new food items such as restructured meat and seafood, new textured sausages and fish cakes, retort resistant soybean curd, Iong-life noodles, volume-improved bread, etc. We present here an overview of developments in microbial TGase technology to date.
A three-dimensional (3D) visualization technique was developed to visualize the distributions of protein and starch inside the whole body of a brown rice grain. The automatic precision microtome system that was developed for microscopy was applied to obtain sections from the whole body of a single grain. The obtained sections were sequentially adhered onto a specialized adhesive tape for 3D reconstruction. All sections were stained by a chemical technique to visualize the protein and starch. Each stained section was digitally imaged using a color charge-coupled device (CCD) camera. The two-dimensional (2D) digital images contained the color information representing protein and starch. A 3D image of the whole body of a brown rice grain was reconstructed by accumulating the 2D digital images in a personal computer. A three-dimensional representation of the distributions of protein and starch in a grain of brown rice was thus constructed.
A temperature distribution study was carried out in an industrial scale static retort during steam sterilization of cans packed with tuna fish. Interest was focused on the surface temperature of conduction heating foods in cylindrical cans placed at various positions in the retort. Since saturated steam release latent heat at a temperature which is governed by pressure and has a large heat transfer coefficient, temperature at the outside surfaces of cans to which it is in contact with might be regarded as the same from one can to another. On the otherhand some process engineers may have experienced that there is a non-uniform temperature distribution in the retort during steam sterilization. Unfortunately, however, no data have been published in literature. In this paper, temperature on the trunk surface of 23 cans selected from a pack of 570 cans loaded into a commercial retort was observed during steam sterilization at 113°C for 80 min including come-up time. From the monitored data, it was observed that, considerable deviations in temperature took place during the come-up period (9 min) but it converged to a constant thereafter. A maximum of 40°C difference in temperature and the delay of 3 min in temperature rise was observed during the come-up period. This deviation at the early stages of heating was found to affect 30% change in sterilization value even though the come-up period was so short less then 11% of the total heating time.
The inactivation behavior of four kinds of bacterial spores by thermal treatments combined with high hydrostatic pressure (HHP) were investigated in a temperature range of 35-120°C and in a pressure range of 0.1-400 MPa. The survival curves of each species of spores became linear at 0.1 MPa, but indicated different time courses by the treatments. The survival curves of B. subtilis and B. stearothermophilus spores became convex downward in the treatments, whereas those of B. coagulans and C. sporogenes spores were linear. Each spores did not die by heating below 100°C at 0.1 MPa. However, the HHP thermal treatments below 100°C quickly killed the bacterial spores except for the B. coagulans. Some of the B. stearothermophilus spores remained alive even at 120°C for 50 min under 400 MPa, whereas the B. stearothermophilus spores surviving at 120°C under 400 MPa started to die when the pressure was released to 0.2 MPa.
Production of γ-aminobutyric acid (GABA), which shows an antihypertensive effect, from glutamate was investigated using rice germ or bran as enzyme (glutamate decarboxylase; GAD) sources. GAD activity in the rice germ was dependent on the concentrations of glutamate and pyridoxal phosphate. The maximum activity in the germ was estimated to be 8.8 μmol/min/g. By a pH-controlled batch reaction using the rice germ, we produced 29.0 g of GABA per 100 g of the germ by incubation for 6 h at 40°C, with the conversion rate of 87.9% . The rice bran was also an effective catalyst for GABA production. These results suggested that our methods are efficient for manufacturing GABA, which is useful in the development of functional foods to manage hypertension.
An inverse procedure has been proposed for on-line determination of thermal parameters in conduction heating foods to achieve appropriate sterilization. The proposed method made use of data collected at the early stages of the heating phase to predict the parameters responsible for the transfer of heat in the conduction heating food. Inherent with the proposed method are some limitations and instabilities, which are manifested especially when there happened to be noise or disturbances during data acquisition. To eliminate these limitations and instabilities a new robust procedure for on-line control to achieve appropriate sterilization in conduction heating foods is proposed in this paper. This method makes use of the well known fact that after a sufficiently large amount of time has elapsed, a semi-logarithmic plot of temperature against time yields a straight line: of which, from the slope and intercept, the thermal parameters responsible for heat transfer are determined. The inflection point in the time/temperature curve is used as an index to detect the time from which the straight line commences. The use of estimated parameters was validated by experiments to be helpful to achieve a targeted lethality.
Linoleic acid was encapsulated with gum arabic, pullulan or maltodextrin at various weight ratios of the acid to wall materials by freeze-drying, and its autoxidation and solvent-extraction processes were measured. Linoleic acid encapsulated with pullulan or maltodextrin at any ratio was highly resistant to autoxidation. When gum arabic was used as the wall material, Iinoleic acid encapsulated at smaller ratios was more easily oxidized. On the other hand, linoleic acid was almost totally extracted with chloroform within scores of minutes of soaking time without any dependence on the wall material or the weight ratio.
An extracellular cysteine protease inhibitor (ECPI) from Chlorella sp. was purified to homogeneity by DEAE-Cellulose column chromatography and Sephacryl S-300 column chromatography. The molecular mass of the inhibitor was estimated to be 284 kDa by SDS-PAGE and 285 kDa by gel filtration on Sephacryl S-300 column chromatography. ECPI retained 100 % of its original activity even after heating at 100°C for 20 min. It showed an inhibitory activity against the proteolytic activity of papain, ficin, chymopapain, calcium activated protease (calpain) and bromelain, but not against trypsin or α-chymotrypsin. ECPI contained 83.4% carbohydrate residues by weight and inhibited papain at a ratio of 1 : 1. The inactivated inhibitor, treated by sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (2-ME), was reactivated by keeping it at 10°C and pH 8.5 after elimination of SDS and 2-ME.
Previous studies have demonstrated antitumor activity in squid ink. This study was carried out to clarify the key component of, and the role of tyrosinase in the antitumor activity. The antitumor fraction, which contains illexin-peptidoglycan, soluble melanin and tyrosinase activity, was isolated from the defatted ink using Tris-HCl buffer extraction, DEAE Sephacel and Sephacryl S-300 chromatography. During the isolation procedure, the behavior of the tyrosinase activity exhibited the same pattern as the antitumor activity. The antitumor fraction could be separated by Phenyl Sepharose CL-4B chromatography into three fractions: illexin-peptidoglycan, tyrosinase and the complex of the two. The fraction containing illexin-peptidoglycan and tyrosinase showed the highest activity against the Meth A tumor in BALB/c mice. This suggests that both these components are needed for the antitumor activity of squid ink.
The antitumor activities of a 70 kDa highly glycosylated fragment (OVMα70F) in the α-subunit separated from pronase-treated hen egg white ovomucin were examined in a double grafted tumor system. BALB/c mice received simultaneous inoculations of Meth-A tumor cells in the right flank (1×106 cells) and left flank (2×105 cells). Solutions (250 μl) of OVMα70F (1000 μg/mouse/day) or physiological saline (PS) were injected into the right tumor on days 3, 4, and 5, and the mice were raised for 21 days. OVMα70F induced direct and almost complete inhibition of growth of the right (treated) tumor and acted indirectly and moderately on growth of the left (distant), compared with each control tumor. The microscopic observations of tumor tissues on day 21 showed that a greater portion of the OVMα70F-treated right tumor cells was necrotic, whereas a smaller portion of the left one was degenerative. In the tumor tissues of OVMα70F-treated mice neutrophils, macrophages and lymphocytes were found to have massively accumulated and the angiogenesis (the formation of new capillary blood vessels) was inhibited.
Variegatic acid (3,3',4,4'-tetrahydroxypulvinic acid, VA) which causes a blueing phenomenon in mushrooms was isolated from Boletus subvelutipes. B. subvelutipes contains 916 mg VA/100 g dry weight of the fruit body. A mixture of VA and acetone powder from B. subvelutipes caused removal of methyl mercaptan (MeSH) after blueing. Optimum pH for the blueing activity by polyphenol oxidase from B. subvelutipes was 5.0 and a high deodorizing activity was observed around the optimum pH. The rate of MeSH removal with VA in 0.4 M potassium phosphate buffer (pH 8.0) was about 36 times as high as that with (−)-epigallocatechin gallate, which has been reported to have the highest deodorizing activity among tea catechins. We isolated several kinds of conjugates of VA molecules with 1-3 MeSH molecules. These results suggest that VA has a high deodorizing activity because it can easily be oxidized and can bind to MeSHs at 6 positions in the 2 dihydroxybenzene rings of VA.
The purpose of this study was to efficiently and enzymatically synthesize the highly unsaturated fatty acid (HUFA) ethyl esters from fish oils and ethanol using an immobilized lipase. Fish oils, ethanol, and immobilized lipases were mixed in n-hexane and then incubated at 30 to 60°C with shaking at 150 rpm for 12 h. Among the immobilized lipases, Lipozyme IM derived from Rhizomucor miehei showed the highest yield of HUFA ethyl esters from fish oils. The optimal temperature for the ethanolysis was between 40 and 50°C. The water added to the lipase in the range of 0 to 10 wt% did not affect the yield of ethyl esters although the presence of over 15 wt% of water induced the hydrolysis. The yield of HUFA ethyl esters from fish oil reached approximately 95% after incubation for 6 h, while those from EPA-25 and DHA-25 were maximal after 12-h incubation. The yields of ethyl esters of EPA and DHA from fish oils were more than 95% and 52-71% after 12-h incubation, respectively. Ethyl esters synthesized from fish oils by an immobilized lipase contained fewer hydroperoxides and conjugated fatty acids than those synthesized by chemical method. This enzymatic method using an immobilized Rhizomucor miehei lipase could be very effective for the conversion of marine oils to their corresponding ethyl esters.
The effect of pH on the inactivation of some enzymes (acid protease, alkaline protease, papain, and glucoamylase) dissolved in McIlvaine buffer by microbubble supercritical (SC) CO2 treatment was investigated in the range of pH 3 to pH 6. Remarkable inactivation was observed with this treatment at pH less than 4, whereas with gaseous and liquid CO2 treatments little or no important inactivation occurred. Microbubbling of SC-CO2 at lower pH was effective at 40-50°C. Microbubbling of SC-CO2 at pH 3 inactivated completely enzymes at temperatures 25° lower than that of the thermal treatment (65-75°C). The degree of inactivation in acid protease increased with increasing ethanol concentration.
Methanol extracts of 98 specimens of the family Labiatae were assayed for their HIV-1 revers transcriptaseinhibitory activity. Potent activity was found in the genera Salvia and Glechoma. The major inhibitory compound in S. officinalis was identified as oleanolic acid, a pentacyclic triterpenoid, by means of 1H-NMR, 13C-NMR, and EI-MS. This compound showed an IC50 value of 1.6-2.0 μg/ml, similar to the authentic oleanolic acid preparation. Oleanolic acid was not a common component of the genus Salvia and not the sole inhibitory compound in Salvia.
Volatile compounds of shiitake mushroom (Lentinus edodes Sing.) samples were extracted by solid phase microextraction (SPME) and then analyzed by GC and GC-MS. Fresh shiitake contained very low level volatile compounds, the major volatile compounds being: 1-octen-3-ol, 3-octanone, dimethyl disulfide and dimethyl trisulfide. During aging, all volatile cornpounds decreased rapidly. Fresh and crushed shiitake contained 6-4000 times more C8 compounds than the uncrushed, the 3-octanone, 1,2,4-trithiolane, and 1-(methylthio)dimethyl disulfide contents increased and/or then decreased during aging. Drying of shiitake mushrooms caused loss of C8 compounds and straight chain sulfur compounds such as dimethyl disulfide, however, it increased the cyclic sulfur compounds 1,2,4-trithiolane and 1,2,4,5-tetrathiane. Contents of 1,2,4-trithiolane and lenthionine in 40°C or 70°C water-soaked dried shiitake increased significantly during soaking. Changes of volatile compounds in fresh or 70°C water-soaked dried shiitake during heating in boiling water were also reported. Contents of lenthionine decreased significantly after heating in boiling water.
The exposure of human lymphoid leukemia Molt 4B cells to sesamol, a component of sesame oil led to both growth inhibition and the induction of apoptosis. Morphological change showing apoptotic bodies was observed in the cells treated with sesamol. The fragmentation of DNA by sesamol to oligonucleosomal-sized fragments that are characteristics of apoptosis was observed to be concentration- and time-dependent. On the other hand, in the sesamol-treated normal human lymphocytes morphological change and DNA fragmentation were not observed at all. These findings suggest that growth inhibition of Molt 4B cells by sesamol results from the induction of apoptosis in the cells.