Enzymotec
`Delivering Lipids
`
`CX—O81 O
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`CX Page 1
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` K-REHL
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`......x
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`NEPN Ex.
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`.1 1
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`Aker v. Neptune
`IPR2014-00003
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`-\
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`/REAL KRILL OIL
`NEP877|TC-00643836
`
`CX-0810
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`CX Page 1
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`>
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`NEP877ITC-00643836
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`NEPN Ex. 2041
`Aker v. Neptune
`IPR2014-00003
`
`

`
`Introduction to MSO™
`Multi Stage Oil Extract ion Process
`
`Introduction
`
` Krill Oil is one of the fastest growing, most
` .sraey tnecer ni stneidergni 3-agemO elbaulav
`Clinical and pre-clinical studies that compare Krill
`Oil and Fish Oil suggest that Krill Oil may be more
`effective than Fish Oil in terms of bioavailability for
`some indications. Although Krill Oil and Fish Oil
`possess a similar name, the term “oil” can be
`misleading not just because it implies a similar
`physical structure but also may suggest the two
`products are being produced and purified using
`similar
`techniques.
` Indeed,
`both of
`these
`perceptions are incorrect and misleading. First
`and
`foremost, Krill Oil
`composition
`is
`fundamentally different than Fish Oil. Unlike Fish
`Oil, Krill Oil contains additional elements within its
`matrix, which seems to enhance its biological
` .slio hsfi ni nwonk si tahw dnoyeb ytivitca
`Krill Oil
`
`Those elements are:
`
`Omega-3 bound Phospholipids - Human clinical
`trials and pre-clinical studies suggest
`that
`phospholipids are better Fatty acids (FA) carriers
`when compared to triglycerides and therefore
`omega-3 FAs attached to Phospholipids are
`considered to have high bioavailability.
`
`Astaxanthin - Recognized as one of the most
`potent natural antioxidants known
`to man,
`astaxanthin is one of the most desirable and
`important components in Krill Oil. Clinical studies
`conducted on Astaxanthin demonstrate
`the
`beneficial effect of this antioxidant on different
`health problems, such as: cardiovascular health,
`immune-related issues, inflammatory and
`neurodegenerative diseases.
`Fish Oil
`
`• Omega-3 Triglycerides
`• Omega-3 Phopholipidis
`• Astaxanthin
`
`• Omega-3 Triglycerides
`
` Fig 1: Schematic composition of Krill Oil vs. Fish Oil
`
`CONFIDENTIAL
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`CX Page 2
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`NEP877ITC-00643837
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`

`
`Introduction to MSO™
`Multi Stage Oil Extraction Process
`
`
`Purification of Fish Oil vs. Krill Oil
`Normally, oil processing for food applications
`(e.g. corn oil, olive oil, palm oil, etc.) involves
`several stages, among which are crushing the
`seed, filtration, deodorization and secondary
`purification processes.To obtain high-quality Fish
`Oil with virtually no contaminants (pesticides,
`polycyclic aromatic hydrocarbons, dioxins and
`polychlorinated biphenyls);
`the purification
`process must include refining and in some cases
`deodorization and distillation stages. The refining
` .tnemtaert lacimehc dna lamreht sevlovni egats
`It is therefore considered a very aggressive
`purification process, significantly affecting the
`quality of the oil and requiring quality parameters
`mainly to control its oxidation level PV, TOTOX
`value, and P-Anisidine - which are therefore
`carefully monitored. Oxidized oil is both not tasty
`and hazardous for your health. In some cases
`even
` considered carcinogenic.
`
`In all matters concerning oil quality, high levels of
`it as
`the above parameters would specify
`adulterated.
`
`It is clear therefore that such a process, though
`efficient, is primarily dependent on exposing the
`biomass to high temperatures and aggressive
`chemicals, especially w hen involving oils.
`The physical character of this cleaning process of
`oils in general cannot be applied in Krill Oil as
`Omega-3 Phospholipids and Astaxanthin are
`extremely sensitive. Furthermore, a different set of
`quality parameters should be implemented as
`TOTOX, PV, etc. are inapplicable. This type of
`process is called Solvent E xtraction, when for
`example the solid biomass is placed in an
`agitated vessel that contains a solvent such as
`alcohol. While mixing the solid particles and the
`solvent, certain elements from the solid particles
`are dissolving in the solvent (for example: dirt in
`soap), while others do not dissolve. The end
`result is a solvent “ loaded” with certain
` selcitrap
`elements and solid elements and solid
` .lessev elgnis a ni detatiga lla
`
`Due to its unique and sensitive components, Krill
`Oil requires specialized knowledge of chemistry
`and process engineering in order to maintain
`appropriate purity, stability, and quality under mild
`processing
`conditions. This procedure
`is
`fundamentally different
`than
`that of Fish Oil
`Triglycerides(TG) purification, normally performed
`under the conditions of high temperature and
`aggressive chemicals.
`
`The purpose of this paper is to explain in simple
`terms the differences in production / purification
`processes of Krill and Fish Oils and to examine
`the
` big impact those processes have on quality
`and
` efficacy of the end product.
`
`Marine biomass processing,
`Fish vs. Krill
`Immediately after death, marine animals begin to
`deteriorate; once autolysis begins, bacteria
`present in the gut and skin or in water can easily
`enter tissues causing further deterioration and
`spoilage. Unfortunately, Krill is more susceptible
`to spoilage and deterioration than other marine
`sources,
`therefore Krill biomass must be
`processed immediately after harvesting on board
`the vessels, while fish processing can be done in
`land-based factories. Therefore, the first stage for
`having quality Krill Oil is the controlled stage of Krill
`biomass preparation. If not properly controlled no
`quality
`Krill Oil
`could
`be
`processed,
`regardless of technology used. Part of the MSO™
`quality pledge, which will be described further, is
`the proper quality
` assurance program which
`ensures the selection of
` the right raw materials
`and suppliers.
`
`CONFIDENTIAL
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`NEP877ITC-00643838
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`

`
`Introduction to MSO™
`Multi Stage Oil Extraction Process
`
`
`The solid particles and the loaded solvent can be
`easily separated through the forces of gravity and
`filtration. T hen a second stage occurs. U nder
`certain conditions (for example: temperature,
`acidity or presence of yet another liquid), the
`dissolved load could be separated from its carrier
`and form a separate phase, sometimes forming
`again solids, which in their turn, must again be
` .noitartlfi dna ytivarg fo secrof hguorht detarapes
`This process, when conducted under controlled
`conditions, can ensure low temperature, zero
`oxidation and high yields. This is generally the
`method utilized by all suppliers of Krill Oil.
`Fig 2 provides a general overview of Fish Oil and
`Krill Oil processing and purification stages.
`
`The following table summarizes the differences
`between quality assays used for Fish and Krill Oil.
`
`As mentioned before, in theory a controlled
`extraction process would ensure a high-quality
`product. B ut in reality, different producers and
`production plants employ various methods with
`different levels of expertise, creating an output
`without standardized results. Therefore a careful
`examination of the output of each and one of the
`Krill Oils currently available shows dramatic
`differences in both qualitative aspects of the oils
`such as: odor, flowability, color and quantitative
`aspects such as levels of oxidation or volatile
`amine-derivatives.
`
`Parameters
`
`Fish Oil
`
`Assay of actives
`
`GC
`
`Krill Oil
`
`P-NMR, GC, HPLC
`
`Oxidation
`
`PV, P-Anisidine, TOTOX
`
`PV, Astaxanthin levels over time
`
`Other qualifying tests
`
`NA
`
`Qualitative spec
`
`Smell
`
`Table 1: Summary of quality essays
`
`TVN, TMA
`
`Smell, Color, Flowability
`
`Fish
`Biomass
`
`Cooking
`& Pressing
`
`Refining /
`Deodorization
`
`Fish Oil
`
`Krill
`Biomass
`
`Cooking
`& Pressing
`
`Extraction
`
`Crude
`Krill Oil
`
`MSOTM
`
`Enzymotec
`K•REALTM
`
`Fig 2: Schematic processing of Fish Oil vs. Krill Oil.
`
`CONFIDENTIAL
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`CX Page 4
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`NEP877ITC-00643839
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`

`
`Introduction to MSO™
`Multi Stage Oil E xtraction Process
`
`
`
`MSO™ vs. Standard Extraction
`MSO™ by E nzymotec is a multi-stage solvent
`extraction pr ocess exclusively developed and
`designed by Enzymotec Engineers specifically for
`Krill Oil extr action and purification. It involves
`careful and controlled steps to assure the highest
`quality, stability and efficacy of Krill Oil.
`
`The Impact of MSO™ on efficacy
`and quality of Krill Oil
`three known
`Generally speaking,
`there are
`parameters used to measure Krill Oil efficacy and
`quality; levels of EPA/DHA, Total Phospholipids
`(PL) and Astaxanthin. Krill Oil is comprised of all
`three of these active elements.
`
`Parameters
`Stability
`
`Smell
`
`Typical Values
`
`% w/w Phospholipids
`
`% w/w EPA+DHA
`
`Astaxanthin (ppm)
`
`40-50%
`
`16-18%
`
`200-400
`
`Table 3: Standard quality parameters for
`Krill Oil
`
`In reality, the term “quality” is broadly used within
`a wide range of definitions and applications.
`
`In the fishing industry the term “quality fish” is
`synonymous with aesthetic appearance and
`freshness and refers to the degree of spoilage the
`fish has undergone. A European Union directive
`on fish hygiene specifies that if the organoleptic
`examination reveals any doubt regarding the
`freshness of the fish, inspectors must use total
`volatile basic nitrogen (TVN) as a chemical test.
`
`TVN is a comprehensive spoilage index that
`includes
` trimethylamine (TMA), ammonia and
`other basic
` nitrogenous compounds.
`
`Parameters
`
`MSO™
`(Krill)
`
`Standard
`Extraction
`(Krill)
`
`Refining
`(Fish)
`
`Temperature
`
`Low
`
`Low
`
`Very High
`
`Stability
`
`High
`
`Poor
`
`Good /
`Standard
`
`Smell
`
`Good /
`Standard
`
`Poor
`
`Good /
`Standard
`
`Table 2: A comparison between methods
`of purification.
`
`CONFIDENTIAL
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`NEP877ITC-00643840
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`

`
`Introduction to MSO™
`Multi Stage Oil Extraction Process
`
`
`Standard list of TVN values for different degrees
`of freshness of fish is as follows:
`
`Parameters
`
`TVN [mgN/100gr]
`
`As previously shown, higher TVN levels
`represents a degraded marine bio-mass.
`Uncontrolled extraction may cause Krill Oil to
`further deteriorate after processing and
`encapsulation.
`
`Standard Extraction
`MSO™
`
`Acceptability Threshold
`
`Time
`Fig 3b: MSO™ impact on Bio-activity as
`measured by Total Volatile Nitrogen (TVN)
`
`Conclusion
`
`While public awareness, of Omega-3 in general
`and Krill Oil in particular, is constantly increasing,
`so is the corresponding need to understand the
`physical and biological differences between the
`various sources of
`these omega-3 oils,
`in
`particular Fish and Krill Oils.
`
`The growing body of knowledge simply
`emphasizes the differences between the types of
`oils and their chemical str ucture, while the
`significant differences between
`the various
`purification techniques and their impact on
`quality and efficacy of the end product is ignored.
`
`Enzymotec defines a new quality level through its
`exclusive MSO™ technology, with improved physi-
`cal properties as confirmed by third party assays.
`These physical properties include decreased
`odor ,increased flow-ability and better biological
`activity (levels of oxidation and amine derivative).
`
`Enzymotec’s MSO™ technology helps overcome
`application challenges and guar antees the
`highest quality standards available in market.
`
`TVN Level (mgN/100 gr)
`
`Fresh Fish
`
`Slight decomposition (edible)
`
`Badly decomposed/non edible
`
`12
`
`12-20
`
`> 25
`
`Table 4: Levels of TVN and the freshness
`degree of fish
`
`Additional quality features and criteria significantly
`affect Krill Oil’s ability to have a positive biological
`
`effect on the body. As will be further demonstrated,
`MSO™ by Enzymotec, does ensure low TVN levels over
`time. Furthermore, careful analysis of certain actives
`over time may also provide an important insight into
`the bio-efficacy of Krill Oil.
`
`MSO™ impact on Bio-activity
`Astaxanthin is a powerful yet sensitive antioxidant. The
`presence of oxidizing elements within Krill Oil quickly
`reacts with the Astaxanthin and degrades it. The figure
`below shows how quickly this can happen when utilizing
`utilizing improper extraction methods.
`
`MSO™
`
`Conventional Extraction
`Processes
`
`% Astaxantin Degradation
`
`Time (Days)
`
`Fig 3a: MSO™ impact on Bio-activity as
`measured by Astaxanthin levels
`
`CONFIDENTIAL
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`CX Page 6
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`NEP877ITC-00643841