Fisheries Industry Promotion Expenses, Fiscal Years 1977—1983
`
`Research & Development for Processing and Usage of
`Marine Products
`
`Comprehensive Report
`
`March 1985
`
`Library of the Ministry of
`Agriculture. Forestry, and Fisheries
`
`
`iiiiiiiiimiiii '
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`Fisheries Agency Research Department, Research Division
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`ENZYMOTEC - 1005 000001
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`Petition for Inter Partes Review
`Of U.S. Patent 8,278,351
`Exhibit
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`[Sealz Library of the Ministry of Agriculture, Forestry, and Fisheries;
`Contributed November 25, 1986;
`
`1 19453]
`
`Table of Contents
`I. General Overview ...............................................................................................................................................................
`
`1
`
`3
`1 Development of technology to make krill edible ..........................................................................................................
`2 Development of technology to make unused fish edible ............................................................................................... 11
`3 Development of technology for using fish processing waste and the like ..................................................................... 21
`4 Development of technology for advanced use of lean fish that can be harvested multiple times ................................. 33
`
`II. Development of Technology to Make Krill Edible ........................................................................................................... 39
`1 Objective basic research for making [various marine products] edible ......................................................................... 4l
`(1) Biochemical properties of krill muscle proteins ............ Hokkaido University, Faculty of Fisheries ..................... 43
`(2) Fractionation of salt-soluble protein .................. Tokai Fisheries Research Institute, Usage Dept. ....................... 50
`(3) Organization of krill proteins ................... Tokai Fisheries Research Institute, Biochemistry Faculty .................. 54
`(4) Technology to recover valuable matter fi‘om krill....Tokai Fisheries Research Institute, Usage Dept................... 65
`(5)
`Improvement of the palatability of krill ............ Tokai Fisheries Research Institute, Usage Dept.......................... 78
`2 Development of technology for mincing of frozen krill....National Fish Paste Marine Processing Cooperative Assn. 89
`3 Development of technology for frozen blocks of krill ...... Japan Joint Whaling Co., Ltd. ............................................ 99
`4 Development of technology to make crab meat-like material from krill ...Ajinomoto Co., Inc. Central Research Inst. 111
`5 Development of technology to separate salt-soluble proteins ....................................................................................... 149
`(1) Technology to separate salt-soluble proteins ............. Ehime Industrial Technology Center ................................. 151
`(2) Development of technology to separate salt-soluble proteins in krill .. Ocean Fisheries Ltd. Ocean Research Ctr. 172
`6 Development of technology to recover valuable matter from krill (astaxanthin) .. NISSUI Central Research Inst. ..... 273
`7 Development of technology for using valuable matter (astaxanthin) ............................................................................ 309
`(1) Use of valuable krill matter (astaxanthin) for fish feed .......... Japan Fish Feed Association .................................. 311
`(2)
`Influence of astaxanthin-containing feed on flesh color, etc., of rainbow trout
`.................. Shizuoka Prefecture Fisheries ExperimentalStation 320
`Influence of astaxanthin-containing feed on body color of red sea bream
`.................. Nagasaki Prefecture Fisheries ExperimentalStation 349
`
`(3)
`
`III. Development of Technology to Make Unused Fish Edible .............................................................................................. 375
`1 Objective basic research for making [various marine products] edible ......................................................................... 377
`(1) Biochemical research pertaining to degradation of freshness in sharks ..... Tokai Fisheries Research Institute ..... 379
`(2) Changes in the process of processing of non-protein — N compounds ....... Tokai Fisheries Research Institute ..... 405
`(3) Characteristics of collagen and gelatin from sharks ...................... National Fisheries University ......................... 417
`(4) Research pertaining to proteins characteristic ot'shark muscle ..... National Fisheries University ......................... 426
`(5) Characteristics of proteins in Macrouridae and shark muscle. . .Hokkaido University, Faculty of Fisheries ........ 434
`(6) Composition and gelling properties of proteins characteristic of shark muscle
`443
`.......................................................................................... Kagoshima University, Faculty ot'Fisheries
`(7) Basic research pertaining to non-glyceride lipids ot'deep-sea fish ..... Hiroshima University ................................ 468
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`(8) Analysis of heavy metals in sharks and Macrouridae ............ Tokyo University of Fisheries ............................... 485
`2. Development of technology for commercialization of frozen mincemeat / paste ......................................................... 493
`(1) Frozen storage conditions and paste production suitability of shark meat
`............................................................................................... Kagoshima University, Faculty of Fisheries.......... 495
`(2) Development of technology pertaining to frozen mincing of deep-sea demersal fish
`............................................................................................... Kushiro Fisheries Experimental Station
`(3) Development of technology for frozen mincing of shark species
`.................................................................... National Fish Paste Marine Processing Cooperative Association 540
`(4) Development of mass production technology for frozen mincing of deep-sea Macrouridae species
`.................................................................... National Surimi [Minced Fish] Manufacturers Association 555
`
`525
`
`IV. Development of Technology for Using Fish Processing Waste and the Like .................................................................. 563
`1. Development of technology for fucoidin—based natural flocculant production. Kimitsu Chemical Industry Co., Ltd. 565
`2. Objective basic research for the purpose of using scum ............................................................................................... 587
`(l) Insolubilizing of water-soluble proteins through pH adjustments ............... National Fisheries University ........... 589
`(2) Dehydration and degreasing of scum ....................................... Tohoku University, Agricultural Department ...... 596
`(3) Research on safety of scum through polymer flocculants (Heavy metal and polymer flocculant monomer analysis)
`...................................... Tokyo University ofFisheries
`608
`3. Recovery of scum from wastewater and technology for treating it .............................................................................. 621
`(1) Development of recovery of flocculant (scum) from wastewater using natural coagulants and treatment technology
`for it ............................... Miyagi Prefectural Marine Product Processing Laboratory ............................................ 623
`(2) Recovery technology for lipids and proteins from wastewater
`651
`................................................................................. Nagasaki Prefecture Fisheries Experimental Station
`(3) Development of technology for the recovery of fish oil and fine particles of fish meat in wastewater, along with the
`development of recovery and degreasing technology for scum
`.................................... Nagasaki Fisheries Processing Cooperative Assoc1ation 670
`(4) Development of usage techniques and degreasing technology for scum
`................ Kushiro Fisheries Experimental Station, Kushiro Municipal Fisheries Processing Cooperative Assn. 697
`(5) Development of an efficient treatment system in order to recover useful matter from wastewater produced in lean fish
`mincemeat that can be harvested multiple times .................... Nitto Chemical Industry Co ..................................... 714
`4. Development of technology for using scum ................................................................................................................. 733
`(1) Research pertaining to the nutritional value and safety of recovered scum
`.......................................................................... Tokai Fisheries Research Institute, Usage Dept. 735
`(2) Study of the safety and nutritional value of scum compared to chicken
`.......................................................................... Tottori Prefecture Food Processing Institute............................... 742
`(3) Value of scum as feed compared to chicken ..... Japan Scientific Feeds Association ............................................. 755
`(4) Component analysis and material property testing in order to use scum for fish feed
`.......................................................................... Japan Fish Feed Association, Oriental Yeast Co., Ltd. 764
`(5) Study of the nutritional value and safety of scum against rainbow trout and eel
`781
`.......................................................................... Shizuoka Prefecture Fisheries Experimental Station
`(6) Development of technology for using scum and excess sludge (Testing pertaining to the decomposition of oil in
`scum by microorganisms, along with the composting of scum and excess sludge by fermentation treatment)
`.......................................................................... Morinaga Engineering Co., Ltd.
`
`816
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`V. Development of Technology for Advanced Use of Lean Fish That Can Be Harvested Multiple Times .......................... 833
`1. Research pertaining to the improvement of the processing suitability of Japanese pilchard by short-term farming
`.......................................................................... Ishikawa Prefecture Fisheries Experimental Station................... 835
`2. Implementation testing of decompressed [exposure] ....National Surimi [Minced Fish] Manufacturers Association .. 851
`3. Recovery of water-soluble proteins through membrane separation technology
`.......................................................................... Ishikawajima-Harima Heavy Industries Co., Ltd.
`4. Technology for using membrane-concentrated water-soluble proteins
`.......................................................................... Sophia University, Faculty ofScience and Technology
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`I. General Overview
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`6. Development of technology for recovery of valuable substances
`(astaxanthin) from krill
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`Development of technology for recovery of valuable substances (astaxanthin) from krill
`
`ASSIGNED TASK
`
`Nippon Suisan Kaisha, Ltd. Central Laboratory — Takao FUJITA
`
`ORGANIZATION AND PERSON IN CHARGE
`
`Fiscal year 1980 to fiscal year 1983
`
`DEVELOPMENT PERIOD
`
`RESEARCH AND DEVELOPMENT OBJECTIVES
`
`Raw and boiled whole frozen product, raw Shelled product, meal and the like are produced by Japan’s
`krill fishing industry, but the utilization status of these products cannot necessarily be said to be adequate,
`and the development of new effective methods of utilization has been sought.
`Marine product processing and utilization technology research and development contractors dealing
`with krill utilization have mainly studied the utilization of krill protein in the form of paste, shelled block
`crab-like food product material and the like, but the utilization of other active ingredients is also an
`important research task.
`The objective of this research is to investigate the development of recovery technology and utilization
`in food coloring and feed coloring of the red carotenoid pigment astaxanthin as a trace valuable substance
`in krill lipids as a means of promoting the effective utilization of krill.
`In food coloring,
`in recent year, tougher restrictions have been introduced on tar-based synthetic
`colorants from the standpoint of safety, and demand for natural colorants has increased. This tendency has
`been especially strong when it comes to red coloring, and various natural red colorants such as carotenoids,
`anthocyanins and anthraquinones are being used, but the properties of these materials have advantages and
`disadvantages with regard to color tone, flavor, dyeing affinity, light Stability and heat-resistance. Currently,
`these materials are used according to their appropriateness in various types of food products, but the
`choices that are available are not necessarily sufficient, and there is demand for new natural colorants. I)
`Also,
`in order to improve the color of the body and meat of red sea bream, silver salmon, young
`yellowtail and tiger shrimp or the like, spirulina or synthetic carotenoids such as apocarotenate ester and
`cantaxanthin are used as colorants for feed for farmed fish. However, frozen feed of neomysis or krill is
`used for fish species that require astaxanthin or that show beneficial metabolic effects through the use of
`this feed. 2)
`
`Currently, there are issues with frozen feed such as usability and environmental pollution, and there is
`an increasing tendency to switch from frozen feed to formula feed, particularly pellet feed. There is a
`corresponding demand for astaxanthin as a feed colorant that can be used for this type of feed.
`Approximately half Of the pigment
`in krill
`is present within the krill
`internal organs, with the
`remaining pigment found within the eyes and Shells. Though no pigment is contained within the muscles 6),
`the muscles can be utilized as raw shelled food product x), while the pigment recovered from the byproduct
`shells and internal organs can be utilized as colorant for food or feed. Further, if the pigment recovery
`residue could be utilized as food product or feed, it would be possible to reach the goal of overall effective
`utilization of krill, and this could contribute to the move towards using krill as a food product, which is a
`fundamental topic in this project.
`Therefore, the objectives of this research were to investigate the utilization of the pigment recovery
`residue, and to investigate the methods of ensuring the overall effective use of krill,
`including the
`economics of these methods.
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`RESEARCH METHODOLOGY
`
`Technology to recover and utilize the red pigment astaxanthin from krill was researched. Table 1
`shows an overview of the test parameters and schedule.
`
`
`Table 1 Method of researching the recovery of valuable substances (astaxanthin) from krill
`
`1980
`1981
`1982
`1983
`
`Manufacturing tests
`Extraction method
`
`< Oil extraction test >
`
`4— Manufacturing tests using an intermediate extractor —>
`< Manufacturing tests using an Oil mill pilot apparatus >
`< Paltial purification
`< Concentration
`< Deodorization
`tests >
`tests >
`tests>
`
`Purification method
`
`Utilization tests
`
`Food colorant
`Feed colorant
`
`< Utility tests >
`< Addition tests >
`< Property tests >
`4—Provision of samples for breeding tests —>
`<— Pellet manufacture, property tests —>
`
`Defatted meal 4— Property, utilization tests—>
`
`In order to prepare a large quantity of samples for use in the various utilization tests, a bench-scale
`extractor and refining apparatus were installed within Work Building NO. 2 of the Central Laboratory at
`Nippon Suisan Kaisha, Ltd.
`Also, in order to ensure the effective progress of the tests at the practical level, part of the tests were
`conducted as joint research with various farmers and specialized manufacturers of oil, food colorants and
`feed.
`
`1. Manufacturing test methods
`(1) Extraction tests
`a. Thermal Oil extraction tests
`
`Astaxanthin is a lipid-soluble colorant, and it can be recovered through dissolution in fat.
`However, these tests were performed in order to also look at enzymatic treatment for oil extraction
`using the normal heated centrifugal separation method.
`b. Solvent extraction tests
`
`Solvent extraction raw materials: Krill meal was primarily used as the raw materials for the
`solvent extraction. The testing was performed using commercially-available meal
`that was
`manufactured at a trawler De Laval-type meal plant and then transported within a freezer. The
`amount Of astaxanthin within krill meal tends to decrease during storage and transport, and storage
`tests with the addition of antioxidants were conducted.
`
`Also, the content of astaxanthin within krill eyeballs is extremely high 6‘ g), and therefore,
`shipboard tests on a prototype eyeball separation and collection apparatus were performed under
`the supervision of the National Food Research Institute.
`Solvent extraction conditions: Hexane, ethanol and acetone were primarily used in the
`investigation as these are organic solvents that can be legally used in food products, but
`in
`consideration ofutilization in feed as well as food, solvents such as methanol and isopropanol
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`were also used to study the astaxanthin extractability and the characteristics of the extracted
`pigment oil. Hexane is commonly used in the extraction of edible oils and fats, but in this testing,
`the hexane was mixed with ethanol, or the water content of the raw material meal was adjusted in
`order to investigate the possibility of improving the extractability of the astaxanthin.
`Manufacturing tests using an intermediate extractor: The astaxanthin extraction testing was
`performed using a dip/agitation-type intermediate extractor (Figure 1) that was installed for the
`purposes of this research.
`
`(D Extraction can: SUS 304, buff 300 finish, 300 L
`capacity, stirring speed 0 ~ 120 rpm, with a reflux
`condenser
`
`(3 Filter: SUS 304, buff 300 finish, 180 L capacity,
`filtration area 0.54 m2, with condenser
`6) Primary concentration can: SUS 304, buff 300 finish,
`340 L capacity, stirring speed 18 ~71 rpm, with
`condenser
`
`GD Secondary concentration can: SUS 304, buff300
`finish, 60 L capacity, stirring speed 17 rpm, with
`condenser
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`® Recovered solvent tank: SUS 304, buff 300 finish,
`280 L capacity (x 2)
`© Cleaning solvent tank: SUS 304, buff 300 finish, 154
`L
`
`L/min
`
`(D Extraction liquid tank: SUS 304, buff 300 finish, 160
`L (x 2)
`Diyer: SUS 304, buff 300 finish, 269 L, stirring
`speed 10 rpm, heat transfer area 1.2 in2
`® Vacuum pump: Wet type, 25 NV, 0.75 kW, 80 L/min
`® Filtrate pump: Internal gear OG-M, 0.75 kW, 10
`L/min
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`® Solvent pump: Non-sealed YH-211, 0.75 kW, 30
`
`Extracted oil
`
`Figure 1 Dip/agitation-type intermediate extractor
`
`The manufacturing conditions were investigated, and the samples were purified for use in the
`utilization tests. The method of the extraction test involved the extraction of 40 kg of krill meal
`within the extraction can (capacity of 300 L) using reflux and heating with agitation (50 rpm)
`using an amount of extraction solvent that was 5 times greater than the amount of meal. Then, the
`material was filtered under reduced pressure in the filter, and the residue was washed three times
`using an equivalent amount of solvent. The extracted solution and the wash solution were
`subjected to solvent distillation in the primary concentration can (200 Torr, 30°C) and the
`secondary concentration can (40 Torr, 70°C) in order to obtain the crude pigment oil containing
`astaxanthin. The extraction residue was dried (400 Torr, 80°C) after blowing steam onto the
`material within a dryer in order to remove the solvent and water content, and to obtain the defatted
`krill meal.
`
`Manufacturing test using a once-through intermediate extractor: Extraction using the above-
`noted dip agitation method is a (batch) method using a single extraction can. However, the method
`is that is more commonly used with actual apparatus in the oil manufacturing industiy is the
`countercurrent method, in which extraction is performed using sequentially thinner extraction
`solutions, starting with a concentrated extraction solution. This method involves the use of a
`battery extractor in which the solvent
`is moved in a counterflow, without moving the raw
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`materials, or the use of a rotocel extractor in which both the raw materials and the solvent are
`moved continuously. 10) Because these methods are once-through methods in which the solvent is
`flowed down over a fixed raw material layer, the intermediate test apparatus for once-through
`extraction shown in Figure 2 was used in order to perform large-scale extraction tests to determine
`whether or not the method could be used in the actual apparatus. In other words, the extraction can
`(capacity of 640 L) was filled with 200 ~ 300 kg of krill meal to a height of 75 ~ 100 cm, and the
`material was extracted by flowing solvent at 50 ~ 60°C over the material at a flow rate of 500 ~
`1000 L/h. The eluate was placed in a micellar tank (3000 L) in order to recover the solvent at 65 ~
`75°C and 20 Torr, and then the recovered solvent was flowed into the extraction tank for use in
`extraction. Part ofa battery extractor from Hokoku Corporation was used in this apparatus.
`Vacuum: 20 Torr
`
`® Extraction can: 640 L, with 100-mesh
`filter
`
`(9 Cartridge filter, 10 u
`
`® Micellar tank, 3000 L
`(3 Condenser
`GD Hexane tank
`
`Figure 2 Once-through intermediate extractor
`
`(2) Concentration, purification tests
`Depending on the application, such as whether the material is to be used in food or in feed, it may
`be necessary to decrease the viscosity of the crude pigment oil, to increase the pigment concentration,
`or to purify the material through deodorization or the like. Therefore, the following concentration and
`purification processes were performed.
`Acetone fractionation: Acetone fractional purification was performed using the following normal
`conditions and an intermediate extractor for the purpose of removing the complex lipids present in
`large quantities within the crude pigment oil. 40 kg of crude pigment oil was added to 200 L of acetone
`while stirring the material, and after stirring the solution for 30 minutes at -5°C ~ 20°C, it was left to
`sit at the same temperature. The acetone-insoluble fraction in the lower layer was removed, and the
`acetone-soluble part in the upper layer was subjected to solvent distillation for use as the pigment oil.
`Adsorption column chromatography: Purification was performed via silicic acid or silica gel
`adsorption column chromatography.
`The apparatus that was used was a stainless steel column with an inner diameter of 21 cm and a
`height of 60 cm, which was connected to a high-throughput fraction collector FRAC-300 (with
`detector, Pharmacia).
`Other purification tests: As methods for pigment concentration that are more suited for industrial
`use than column chromatography, urea addition following saponiflcation treatment, low-temperature
`fractionation, and molecular distillation following lipase treatment or separation with metal soap were
`investigated. Also, molecular distillation and reduced pressure steam distillation were performed for
`the purpose of deodorization. The apparatus that were used were a multi-stage stripping tower (Kansai
`Chemical Engineering Co., Ltd.) and a centrifugal-type molecular distillation apparatus MS-150
`(Nippon Sharyo Ltd.)
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`2. Utilization tests
`
`(1) Food pigment
`The tests on the utilization of krill pigment as food pigment were performed using the purified
`pigment oil samples that had undergone concentration and deodorization in order to investigate the
`following parameters.
`light stability, heat
`a. As the basic characteristics of food pigment, the color tone, shelf life,
`resistance, metal resistance and dyeing properties were investigated using oily samples, or using
`samples of water dispersion using an emulsifier.
`b. The suitability for utilization in various types of food products was investigated by adding
`pigment oils with different degrees of purification in order to color various food products, such as
`processed fish cake as an example ofa fishery protein food product, rice crackers as an example of
`a carbohydrate confectionary, and butter cream as an example of an oil and fat food product, and
`then looking at the relationship between taste, color and the degree of purification.
`c. The practicality of use as a food pigment was evaluated by preparing prototypes of colored food
`products such as canned crab sticks and dried salmon flakes.
`The testing on the characteristics and utilization of the material as a food pigment was
`performed as joint testing with San-Ei Chemical Industries Co., Ltd.
`
`(2) Feed pigment
`Feed pellets and feed oil with specified concentrations of astaxanthin were prepared for use in
`tests on red sea bream and rainbow trout feed, and this feed was provided to the Nagasaki Prefecture
`and Shizuoka Prefecture Fisheries Experiment Stations. The colored pellets were prepared by Oriental
`Yeast Co., Ltd.
`The feed oil was provided to the red sea bream and rainbow trout in actual dosing tests performed
`by the farmers.
`Also, krill pigment oil was provided to young yellowtail (Kochi Prefecture Fisheries Experiment
`Station) and silver salmon (Miyagi Prefecture Fisheries Experiment Station) as tests to improve the
`coloring of various fish species.
`The feed oil, colored pellets and krill meal were stored for 3 ~ 6 months in dark locations at
`temperatures of 5°C and 30°C in order to compare the stability of astaxanthin during storage.
`
`(3) Defatted meal
`The effective utilization of defatted krill meal, which can be obtained as a solvent extraction
`residue, is an issue that can greatly affect the cost of krill pigment, and the suitability of the utilization
`of this meal in food products and feed was investigated.
`As utilization tests, rainbow trout nutrition tests (Shizuoka Prefecture Fisheries Experiment
`Station) and systematization tests using an extruder (National Food Research Institute) were performed.
`Also, testing to develop applications, such as testing prepare flavoring agents through hydrolysis and
`palatability tests using farmed fish or pets were performed jointly with related companies.
`
`3. Analysis methods
`Total carotenoids.: According to the methods of Tsukuda 3) and Kanemitsu/Aoe 4), extraction was
`performed using an acetone or chloroform/methanol mixture (2:1), after which ethyl ether solvent transfer,
`water washing and anhydrous sodium sulfate dehydration were performed. The maximum absorbance
`F; 1‘
`>965” 1 9am
`‘
`1 ”ll
`" was used to determine the
`
`within benzene was determined, and the absorption coefficient
`total carotenoids.
`
`the carotenoid
`After development using astaxanthin: silica gel G-60 thin layer chromatography,
`composition was determined using a Shimadzu dual-wavelength chromatoscanner CS-900 (measurement
`wavelength: 480 nm). The total for the composition ratios of the astaxanthin diester, astaxanthin monoester
`and the free astaxanthin was treated as representing astaxanthin, and this composition ratio was multiplied
`by the above-noted total carotenoid amount for use as the astaxanthin amount. The absorption at 2 ~ 3
`spots” observed before and after the astaxanthin monoester on the thin layer chromatogram and the
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`absorption at the origin were measured for use as other carotenoids.
`Total lipids / lipid composition: The total lipids were extracted using FOLCH chlorofonn/methanol,
`and the lipid composition was measured using an Iatroscan TH-lO (Yatron Corporation) following
`development using chloroform, methanol and water (60:35:5).
`Fatty acid composition: Following methylation using boron trifluoromethanol,
`analyzed using a Hitachi 063 gas chromatograph (filler: DEGS, detector: FID).
`Crude protein: The total nitrogen obtained as a result of using a kel checker (Anritsu Electronics K)
`was multiplied by 6.25.
`Crude fiber: The ash content was subtracted from the dry weight of the residue of extraction with
`1.25% NaOH and 1.25% H2504.
`Ethoxyquin: Following acetonitrile extraction, the material was measured at an excitation wavelength
`of 365 nm using a fluorophotometer (Kotaki MKW 155).
`Ignition residue, heavy metals,
`arsenic: The analyses were conducted using the
`Pharmacopoeia General Test Methods.
`Color: The values for L, a, and b were measured using a color-difference meter ND-101DC (Nippon
`Denshoku Industries Co., Ltd.) or a tint/color-difference meter CR-IOO (Minolta Camera C0,, Ltd.).
`
`the material was
`
`Japanese
`
`RESULTS AND DISCUSSION
`
`1. Manufacturing tests
`(I) Study of pigment recovery methods
`a. Oil extraction tests
`
`It has been shown that 3 ~ 4 mg/ 100 g of carotenoids are contained within krill, with the
`primary constituents being astaxanthin diester, astaxanthin monoester and free astaxanthin. 5‘ 6)
`Astaxanthin is a lipid-soluble pigment, and it is most preferable to be able to use the normal
`boiling-out method for fish oil, such as to separate and collect the oily component containing the
`astaxanthin via centrifugal processing of stick water during meal manufacture. However, no oil
`content separation was observed during the normal heating and centrifugal separation conditions.
`This was speculated to be due to the fact that the phospholipids within the krill lipids formed a
`strong emulsion with the protein. Therefore, with the goal of breaking down this emulsion, the
`addition of the following types of treatment into the heating (95°C, 10 minutes) and centrifugation
`(10,000 g, 20 minutes) operations was investigated.
`In other words, experiments were performed involving combinations of various treatments
`such as autolysis, protease (bioprase, pepsin, papain) or lipase (olipase) enzyme treatment,
`acid/alkali decomposition, salt addition,
`low-temperature heating (65°C, 5 minutes), high-
`temperature heating (120°C, 20 min), ultrasonic processing and ultracentrifuge processing, but no
`clear oil content separation was observed.
`
`b. Raw materials for use in solvent extraction
`
`It was judged that, at the current point in time, it would be difficult to perform shipboard
`recovery of astaxanthin via oil content collection, and therefore, it was believed that it would be
`appropriate to perform oil content extraction using the following organic solvents on land, and
`first, the raw materials for extraction were investigated. Table 2 shows various krill products and
`the total carotenoid content within the test specimens.
`The meal used was krill whole meal (Nippon Suisan Kaisha, Ltd, product code M Meal) from
`a trawler De Laval-type meal plant.
`The shell meal was meal manufactured by mixing whole meal with part of the internal organs
`and shells derived from the manufacture of raw shelled product using a roll separator (Nippon
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`

`Suisan Kaisha, Ltd., product code K Meal), and current production volumes for this product are
`extremely low.
`
`
`Table 2 Total carotenoid content (mg) in krill products
`Within 100 g of wet Within 100 g of dry
`wei ht
`wei ht
`
`Within 1 g of lipids
`
`Raw frozen
`
`Boiled frozen
`
`Meal
`
`511611 mfial
`
`Boulton shell test specimens
`
`Test specimens containing
`
`primarily eyeballs
`
`1. 4
`
`1.- 3
`
`1. 3
`
`1,. 3
`
`.1.
`
`11
`
`4, ?
`
`
`
`4. 23
`
`4g I
`
`2 1. 3
`
`1 S. E
`
`5, 1
`
`3 g, 5
`
`2 II). E
`
`1
`
`‘1‘. E
`
`3 E 4
`
`1
`
`9,. 3
`
`2 fl. 2
`
`1
`
`1 3
`
`The Boulton shells were test specimens derived from the boiled shelled product, which
`contain little pigment, and there were issues in terms of securing the raw materials as well.
`The test specimens containing primarily eyeballs were mixtures of small shells and eyeballs
`separated by boiling the boiled krill over metal mesh, and the material contained carotenoids in an
`amount that was approximately five times greater than that observed in the other samples. During
`cruises in 1981, a rotary sieve for use in collecting eyeballs was used to obtain mixtures of
`eyeballs and legs. The carotenoid content in these mixtures was 71 mg/ 100 g within the anhydride.
`While these samples containing a large quantity of eyeballs represent an interesting raw material
`with a high pigment concentration, but at the current point in time, there are issues in terms of
`securing this raw material.
`