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SUMMARY Changes in the lipids of soybeans brought about by Rhizopus orysae during the production of tempeh were studied. The mold possesses strong lipase activity and caused the hydrolysis of over one‐third of the neutral fat of the soybean during the three‐day fermentation. The fatty acid composition of soybean tempeh was compared with that of cooked soybeans by vapor‐phase chromatography of the methylesters. The neutral fat was composed of palmitic, stearic, oleic, linoleic, and linolenic acids, with linoleic acid predominating. These acids were liberated during fermentation in roughly the same proportions found in soybeans after heating 90 min at 100°C. During the most active mold growth, proportionately higher levels of palmitic acid were found, and the level of linoleic acid was somewhat lower. Except for the depletion of some 40% of the linolenic acid in the later stages of the fermentation, there apparently was no preferential utilization of any fatty acid.

This study is focused on the characterization of the interaction of the amphiphilic peptide bombolitin III (from the bumblebee Megabombus pennsylvanicus) with phospholipid monolayers and vesicles. It is shown that due to the amphiphilic character of its alpha-helical conformation this water-soluble peptide is able to interact in an ordered fashion with phospholipid organized structures. Depending on the temperature, the subphase, and the particular phosphatidylcholine used, the mixed peptide-phospholipid monolayers can be homogeneous or display phase separation. This behavior was observed by means of the Langmuir film balance technique, coupled with an epifluorescence microscope. In well-defined conditions it is possible to visualize the formation of phase-separated peptide domains at the air-water interface and to study the effect of their presence on the organization of the lipid. The action of phospholipase A2 at the lipid-peptide interface was also followed by means of fluorescence microscopy: some evidence that the enzyme preferentially hydrolyzes the phospholipid that is in contact with the peptide is presented. Furthermore, the presence of bombolitin III in L-alpha-DLPC monolayers causes an increase in the initial speed of degradation with phospholipase A2. These results are in agreement with previous findings that show that the bombolitins are activators in vitro of phospholipase A2. Experiments were also performed with peptide fragments corresponding to the alpha-helical sequences of the protein uteroglobin: despite some amphiphilic character, these peptides do not interact strongly with phospholipid monolayers. Only one of these peptides (corresponding to the helix 4-14 in uteroglobin) is adsorbed in the monolayer in a similar fashion to bombolitin III but does not cause an increase in the activity of phospholipase A2.

It has been known for a long time that unblanched vegetables deteriorate in quality when held in frozen storage for extended periods of time. Rancidity of the lipid matter is one of the primary causes of off-flavor in unblanched peas which had been stored at 0°F. (-17.8"C.) (5). Large increases in acid numbers together with increases in peroxide numbers were observed in the lipids extracted from raw peas after several years of frozen storage. The main increase in acid number occurred during the first year of storage (8). Similar changes were noted in the lipids extracted from unblanched sweet corn, snap beans, and other vegetables after extended periods of storage at 0°F. (-17.8"C.) ( 4 ) .I n order to extend the knowledge of the changes which take place in such material, the present study was undertaken to determine the length of time that unblanched vegetables could be held in frozen storage before these undesirable changes could be detected, and to determine the course of the development of the acids and peroxides during the period of storage. MATERIALS AND METHODSThomas Laxton peas, Wade snap beans, and Golden Cross sweet corn were grown on the Experiment Station farm and were harvested, processed, and stored, as previously described (4, 5 ) .Quality changes were measured both chemically and by means of a taste panel. Samples from each lot were taken for immediate taste test a t the time of harvest, and at regular intervals during storage, until obvious deterioration in quality was definitely established. A t the same time, samples were dried by 1yophiIization and were extracted for 48 hours with peroxide-free anhydrous ethyl ether to obtain the crude lipid material, as described previously ( 5 ) . This work was started before it was determined that a 24-hour extraction period was sufficient to effect the extraction of the crude lipid (4), but was continued on the 48-hour basis to keep the conditions uniform. The chemical determinations were continued a t regular but lengthening intervals throughout the storage period. A final subjective and chemical examination of the 1952 samples was conducted after 2 years of storage. Chemical methods used were described previously (4, 5).

It is vell known, that to secure high quality frozen peas it is necessary to blanch this vegetable before placing it in storage and that unblanched peas held in frozen storage develop undesirable off-flavors that are frequently described as "hay-like" in character ( 4 ) .The present investigation was started with the object of determining the caiise of these off-flavors. EXPERIMENTAL PROCEDUREPreparation of Sample. Thomas Laxtoii peas were grown on the Experiment Station farm during the 1045 s~a s o n and harvested in the stage that would yield a fancy f roTen product. Thcy were vined mechanically, washed, and hand-picked to eliminate bruised and otherwise injured peas. They were then divided into two lots, one of which was packed without further treatment. The other lot was blanched by immersion in boiling water f o r sixty seconds, cooled in cold water, drained and packed.Both lots were frozen, transferred to storage at --17.8"C.(O0F.) and kept under these conditions for five years. Other lots of this same variety were similarly prepared for immediate analysis during the 1950 season.

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Raw vegetables which are held in frozen storage for extended periods show a progressive decrease in quality, characterized by development of off-flavors.and changes in the composition of lipid matter. One of the major changes occurring during the frozen storage of raw peas and other r a~7 vegetables is the development of peroxides in the lipids (2, 3 ) . There was a loss of chlorophyll and carotene when frozen raw peas were held for periods from one to 6 years at -17.8" C. (3,8,9). I n contrast, blanched peas of the same lots stored under identical conditions did not show this loss of chlorophyll, or changes in coniposition of the lipid materials.The enzyme lipoxidase has been postulated as being a causative agent for chlorophyll breakdown during frozen storage of raw peas (8). The primary action of lipoxidase is to cause a peroxidation of the double bonds of certain unsaturated fatty acids. A concurrent action involves the oxidatioii of certain unsaturated plant pigments such as chlorophyll and carotene (6).To prove that lipoxidase is responsible for peroxidation of lipids and chlorophyll destruction during storage it is necessary, first, to demonstrate its presence in raw peas, and second, this enzyme must be isolated from raw peas, purified, and allowed to react with its substrates in such a manner that the effects of its action may be determined quantitatively.This paper reports the demonstration of the presence of lipoxidase in frozen raw peas, its partial purification, and its action in bringing about the peroxidation of pea lipids and the destruction of chlorophyll in blanched peas.

Enzymes are believed to be responsible for the development of off-flavor in raw and underblanched vegetables during frozen storage (1). A variety of enzymes have been suggested as being causative agents in flavor deterioration (l), but interest has centered mainly around catalase and peroxidase. Tests for the presence of these two enzymes have long been used as criteria of adequacy of blanching of vegetables prior to freezing. Furthermore, a reasonably good correlation has been observed between the presence of residual amounts of these enzymes and extent of off-flavor in frozen vegetables. To date, however, the identity of the responsible enzymes has remained obscure.Recently it has been shown that lipoxidase and lipase are responsible for the progressive deterioration of lipids in raw peas during frozen storage (5, 12). These changes in the lipids, characterized by increases in titratable acidity, development of lipid peroxides, and loss of chlorophyll, closely paralleled the progressive development of off-flavor (7). It was deemed most likely that lipoxidase and possibly lipase are primary causative agents in the production of off-flavors during frozen storage of raw peas.Proof of involvement in off-flavor development of any or all of the above enzymes may be ascertained by a study of their action in model systems. Preparations of catalase, peroxidase, lipoxidase, and lipase of known specific activity were added to enzymatically inert blanched peas, and the samples analyzed after a suitable period of frozen storage. This paper reports the production of off-flavors during the frozen storage of macerates of blanched peas, brought about by the addition of enzymes, and a correlated chemical study of lipid deterioration. MATERIALS AND METHODSPerfected Freezer peas, grown on Experiment Station plots, were harvested and processed as described earlier (5) and stored in 30-pound tins at -17.8" C. The blanched peas to which enzymes were to be added were thawed and blended for 2 minutes in a five-quart Waring blender with one-half their weight of water. The resultant pea slurry was divided into 100 g. lots, and the various enzyme preparations were added and well mixed by stirring. The amounts of enzymes added to blanched peas were calculated to be twice the amounts present in fresh raw peas. Four series of samples were prepared in which the 4 enzymes were added singly and in all possible combinations. Samples of blanched pea slurry with no added enzymes were included as controls in each series. Two series were used for taste test evaluation for flavor and color (I,?), and two series were used for chemical analysis. Samples were placed in cellophane bags inside waxed cartons, frozen, and held in storage at -17.8" C.Catalase activity was measured manometrically by the Thompson method ( I r ) . Crystalline catalase (General Biochemicals, Inc., Chagrin Falls, Ohio), specific activity -0.018 mg. produced 2.5 ml. 0 2 , was added at the level of 17 mg. per 100 g. lot of peas.One gram of the resultant mixture produced 2.8 ml. of ...