DHA-rich Algal Oil from Schizochytrium sp.RT100

36 Pages·2015·1.38 MB·English

DHA-rich Algal Oil from Schizochytrium sp.RT100

A submission to the UK Food Standards Agency requesting consideration of
Substantial Equivalence in accordance with Regulation (EC) No 258/97
concerning novel foods and novel food ingredients
I. Administrative data

Applicant:
DAESANG Corp.
26, Cheonho-daero
Dongdaemun-gu
Seoul 130-706
South Korea
Ray Kim, James Kwak
[email protected], [email protected]
Tel. [phone]

Contact address:
Dr. Stoffer Loman
NutriClaim BV
Lombardije 44
3524 KW Utrecht
The Netherlands
[email protected]
Tel. [phone]

Food ingredient:
The food ingredient for which an opinion on Substantial Equivalence is requested is Daesang Corp.’s
Schizochytrium sp.RT100 derived DHA-rich oil.

Date of application:
20 March, 2015.

1
II. The Issue

In recent years, Schizochytrium sp.-derived docosahexaenoic (DHA)-rich oils have been the subject of
several Novel Food Applications submitted under Regulation No 258/97 in the European Union (EU;
EC, 1997). The first application for Novel Food authorization for Schizochytrium sp.-derived DHA-rich
oil for general use as a nutritional ingredient in foods, was submitted by the United States (US)-based
company OmegaTech Inc. to the Advisory Committee on Novel Foods and Processes (ACNFP) of the
United Kingdom (UK) Food Standards Agency (UK FSA) in 2001.(Martek BioSciences Corporation,
2001)

After evaluation, the placing on the market of OmegaTech DHA-rich oil was authorized in 2003
following the issuing of the Commission Decision of 5 June 2003 (CD 2003/427/EC) authorizing the
placing on the market of oil rich in DHA (docosahexaenoic acid) from the microalgae Schizochytrium
sp. as a novel food ingredient (EC, 2003).

Following the acquisition of OmegaTech Inc. by Martek Biosciences Corp. in 2002, an application
requesting an extension of use for the Schizochytrium-derived DHA-rich oil was filed with the UK FSA
in 2008. The extension of use was authorized by Commission Decision 2009/778/EC (EC, 2009).

In 2011, the Canadian Company Ocean Nutrition Canada Ltd. submitted a request for an opinion on
substantial equivalence of their Schizchytrium sp.ONC-T18 derived DHA-rich oil to that of Martek ‘s
(formerly OmegaTech) DHA-rich oil already authorized (EC, 2003). In March 2012, The ACNFP
concluded that the DHA rich algal oil produced by Ocean Nutrition Canada could be considered to be
substantially equivalent to the existing DHA rich algal oil produced by Martek (ACNFP, 2012).
The current submission pertains to a similar request for an opinion on substantial equivalence of
Daesang DHA-rich oil from Schizochytrium sp.RT100 to that of Martek Biosciences Corp., that was the
initially authorized DHA-rich oil (EC, 2003), including the authorized extension of uses (EC, 2009). As
per 14 July 2014, both Decisions 2003/427/EC and 2009/778/EC have been repealed following the
issuing of Commission Implementing Decision of 14 July 2014 on authorizing the placing on the
market of oil from the microalgae Schizochytrium sp. as a novel food ingredient under Regulation
(EC) No 258/97 of the European Parliament and of the Council (CD 2014/463/EU) and repealing
Decisions 2003/427/EC and 2009/778/EC. Therefore, the current application for an opinion on
substantial equivalence of Schizochytrium sp. DHA-rich oil therefore should be considered to relate
to substantial equivalence to the Specification of oil from the microalgae Schizochytrium sp. as laid
down in Annex 1 to this Commission Implementing Decision as well as the authorized uses of oil from
the microalgae Schizochytrium sp. as laid down in Annex 2 of the Commission Implementing Decision
2014/463/EU (EC, 2014).

2
III. Table of Contents

I. Administrative data ............................................................................................................................. 1
II. The Issue ............................................................................................................................................. 2
III. Table of Contents ................................................................................................................................ i
List of Appendices ................................................................................................................................ii
List of Tables ........................................................................................................................................ii
List of Figures ...................................................................................................................................... iii
1. Introduction .................................................................................................................................... 3
2. Substantial equivalence of the source organism to DSM/Martek’s Schizochytrium ATCC 208884
2.1 Taxonomy and Morphology of Schizochytrium sp. ..................................................................... 4
2.2 Taxonomic Classification .............................................................................................................. 5
2.3 Morphological Taxonomy ............................................................................................................. 6
2.4 Phylogenetic Taxonomy -18S rRNA sequence comparison ......................................................... 7
2.5 Conclusion on substantial equivalence of source organisms .................................................... 12
3. Substantial Equivalence of manufacturing process and characterization of Daesang DHA-rich
oil from Schizochytrium RT100. ............................................................................................................ 13
3.1 Culture conditions of Daesang’s Schizochytrium sp.RT100 ....................................................... 13
3.2 Manufacturing Process of Daesang DHA-rich oil from Schizochytrium sp.RT100 .................... 13
4. Compositional Equivalence .......................................................................................................... 15
4.1 Specification of Daesang DHA-rich oil from Schizochytrium sp.RT100 ..................................... 15
5. Conclusion on substantial equivalence of manufacturing and characterization of product
specifications ........................................................................................................................................ 18
5.1 Equivalence of proximate analysis ............................................................................................. 18
5.2 Equivalence of fatty acid composition ....................................................................................... 19
5.3 Equivalence of nutritional value and metabolism ..................................................................... 21
5.4 Substantial equivalence of Intended Use of Daesang DHA-rich oil from Schizochytrium sp. .. 21
5.5 Substantial equivalence in levels of undesirable substances ................................................... 23
5.5.1 Microbiological information ................................................................................................ 23
5.5.2 Elemental analysis ............................................................................................................... 23
5.5.3 Benzo(a)pyrene .................................................................................................................... 24
5.5.4 Dioxins, dioxin-like PCBs and non-dioxin-like PCBs............................................................ 24
5.5.5 Potential for contamination with Cyanobacteria ............................................................... 25
5.5.6 Potential for contamination with toxin-producing algae .................................................. 26
5.5.7 Prymnesins ........................................................................................................................... 28
i
5.5.8 Hexane residue .................................................................................................................... 29
5.5.9 Conclusion on levels of undesirable substances ................................................................ 29
6. Overall conclusion ............................................................................................................................ 30
7. References......................................................................................................................................... 31

List of Appendices

Appendix A: Full sequence alignment and comparison of 18S rRNA Schizochytrium sp. RT100 and
ATCC20888 …………………………………………………………………………………………………………………………………33
Appendix B: A concise description of the downstream production process .……………………………….35
Appendix C: Test report by Intertek Food Services GmbH .………………………………………………………….37
Appendix D: Test report by Rikilt Institute of Food Safety .………………………………………………………….38

List of Tables

Table 1 18S rRNA sequences of Thraustochytrid Phylogenetic Group I(sites 494-504-shaded
green)- Comparison by strains .................................................................................................... 10
Table 2 18S rRNA sequences of Thraustochytrid Phylogenetic Group II(sites 1712-1739-shaded
green) - Comparison by strains ................................................................................................... 10
Table 3 Daesang product specifications for DHA-rich oil produced by Schizochytrium sp.RT100
according to the above described manufacturing process ............................................................ 15
Table 4 Produc specifications for DHA-rich oil derived from Schizochytrium sp. ........................... 16
Table 5 Compliance to EC specifications of Daesang DHA-rich oil from Schizochytrium sp.RT100
compared with DSM/Martek from Schizochytrium sp.ATCC20888................................................ 16
Table 6 Proximate analysis of Daesang DHA-rich oil from Schizochytrium sp.RT100 and
DSM/Martek from Schizochytrium sp.ATCC20888 ....................................................................... 18
Table 7 Fatty Acid Profiles of Daesang DHA-rich oil from Schizochytrium sp.RT100 and
DSM/Martek from Schizochytrium sp.ATCC20888 with reference of OmegaTech ......................... 20
Table 8 Authorized uses of oil from the microalgae Schizochytrium sp. ....................................... 22
Table 9 Microbiological analysis of Daesang DHA-rich oil from Schizochytrium sp.RT100 and
DSM/Martek from Schizochytrium sp.ATCC20888 ....................................................................... 23
Table 10 Elemental analysis of Daesang DHA-rich oil from Schizochytrium sp.RT100 and
DSM/Martek from Schizochytrium sp.ATCC20888 ....................................................................... 24
Table 11 Levels of benzo(a)pyrene of Daesang DHA-rich oil from Schizochytrium sp.RT100 and
DSM/Martek from Schizochytrium sp.ATCC20888 ....................................................................... 24
ii
Table 12 Contents of dioxins and dioxin-like PCBs of Daesang DHA-rich oil from Schizochytrium
sp.RT100 and DSM/Martek from Schizochytrium sp.ATCC20888 .................................................. 25
Table 13 Algal toxins tested in Schizochytrium sp.RT100 DHA-rich oil .......................................... 27
Table 14 Hexane residues analysis of Daesang DHA-rich oil from Schizochytrium sp.RT100 and
DSM/Martek from Schizochytrium sp.ATCC20888 ....................................................................... 29

List of Figures

Figure 1 RT100 on solid agar, showing successive bipartitioning (arrows) ...................................... 6
Figure 2 Geological Time Periods .................................................................................................. 7
Figure 3 Shared signature sequences of the boxed region (position 494-504) of secondary
structure of 18S rRNA showing close relationships in the thraustochytrid phylogenetic group ........ 8
Figure 4 Shared signature sequences in 18S rRNA showing close relationships in the
thraustochytrid phylogenetic group. The boxed region (position 1712-1739) showing the large
insertion present in all members of thraustochytrid phylogenetic group but absent from all
other organisms ........................................................................................................................... 9
Figure 5 ML trees showing relationships among selected thraustochytrid 18S rRNA sequences.
Bootstrap values are indicated at the nodes. The scale bar at the bottom represents the %
difference between distinct region sequences (e.g. 0.02 = 2%) ..................................................... 11
Figure 6 Set conditions in MEGA5.2 for phylogenetic tree generation ......................................... 12
Figure 7 Flow diagram of DHA-rich oil production from Schizochytrium sp.RT100 ........................ 14

iii
1. Introduction

Docosahexaenoic acid (DHA; 22:6 n-3), a polyunsaturated fatty acid (PUFA), is linked to various
health benefits in humans including cognitive and visual development of infants and reduced risk of
cancer, cardiovascular diseases and mental illnesses of adults. Indeed, recently several health claims
to be made on foods for DHA have been authorized in the EU. These authorized claims pertain to
maintenance of normal brain function and vision and, in combination with eicosapentaenoic acid
(EPA; 20:5 n-3) to contribute to the normal function of the heart.

Traditionally, long-chain polyunsaturated fatty acids (PUFAs), amongst which DHA, are obtained from
marine fish such as salmon, mackerel, and tuna. Fish oil, with an annual production of over 1 million
tons (2010) is at present the major source of DHA. However, heavy metal pollution and over-
exploitation of the sea-fish resources jeopardize the sustainability of this source.

Some marine microalgae such as dinoflagellates and species in the Heterokonta phylum contain a
high amount of DHA. However, the majority of those microalgae are photoautotrophic and as such
being dependent on light as energy source and on weather conditions. Heterotrophic microalgae are
able to take energy from simple organic substances without requiring light (Yokoyama & Honda,
2007). One of heterotrophic microalgae is Schizochytrium sp. which can be utilized as alternative to
fish oils due to its rapid growth rate, its weather condition independency and its DHA content which
reach to almost 49% of its total fat content (Renet al., 2010).

This DHA-rich algal oil is produced by the US-company Martek Biosciences Corp. that obtained
marketing authorization for the product as a Novel Food in the European Union (EU) (EC, 2003). In
2004, the FDA did not object to the GRAS notification by Martek for its DHA algal oil derived from
Schizochytrium sp.(FDA, 2004 -
http://www.fda.gov/Food/IngredientsPackagingLabeling/GRAS/NoticeInventory/ucm153961.htm).
DHA-rich algal oil is now available for use in foods and dietary supplements.

The current application pertains to a request for an opinion on substantial equivalence of Daesang’s
DHA-rich oil from Schizochytrium sp. with the originally authorized (EC, 2003) DHA-algal oil
manufactured by Martek BioSciences Corp.

This submission will be compiled taking the ACNFP guidelines for the presentation of data to
demonstrate substantial equivalence between a novel food or food ingredient and an existing
counterpart into account (ACNFP, 2005). In the following sections it will be argued that based on the
characterization of the source organism Schizochytrium sp.RT100, its method of production, the
composition of the DHA-rich oil, its metabolism, its intended use and level of undesirable substances,
we conclude that Daesang DHA-rich oil from Schizochytrium sp.RT100 is substantially equivalent to
Martek’s Schizochytrium sp.ATCC 20888-derived DHA algal oil.

3
2. Substantial equivalence of the source organism to DSM/Martek’s
Schizochytrium ATCC 20888

2.1 Taxonomy and Morphology of Schizochytrium sp.

Schizochytrium is a heterotrophic microalgae which belongs to the family of Thraustochytriacaea
(Luyinget al., 2008). Schizochytrium is a spherical unicellular microorganism. According to Yokoyama
et al. (2007),morphological characteristics of Schizochytrium under the microscope show ectoplasmic
nets, formation of zoospores, aplanospores, and amoeboid cells of a size between 10-20 μm. The
taxonomy details of Schizochytrium are as follows:

Kingdom : Chromista (Stramenopila)
Phylum : Heterokonta
Class : Thraustochytridae
Order : Thraustochytriales
Family : Thraustochytriaceae
Genus : Schizochytrium
Species : Schizochytrium sp.
(Source: Leipe et al., 1994)

Schizochytrium produces biflagellate zoospores and mature cells divide by repeated binary division to
form diads, tetrads and clusters (Figure 1). Each Schizochytrium cell could develop into a sporangium
that produces several zoospores (Kamlangdee & Fan, 2003).

The Daesang strain was originally isolated from a sea water sample obtained from the nearby sea of
Iriomote island in the Okinawa Prefecture, Japan in 2005 by Prof. Daisuke Honda (Marine
Biotechnology Institute Co., Ltd., Kamaishi, Iwate, Japan, and the National Institute of Bioscience and
Human-Technology, Agency of Industrial and Technology, Tsukuba, Ibaraki, Japan), who is an expert
in the field of marine biodiversity and systematics of marine algae.

Following selection of the strain on the basis of growth characteristics and high content of omega-3
polyunsaturated fatty acids, including docosahexaenoic acid (DHA), further characterization indicated
that the strain belonged to the genus Schizochytrium. The strain has been purchased by Daesang
Corp. and named RT100.

4
2.2 Taxonomic Classification

The Labyrinthulomycota (slime nets, net slime moulds) form a taxonomic phylum consisting of two
families (Olive, 1975). One family (the Thraustochytriaceae) consists of several genera commonly
referred to as “thraustochytrids”.

The traditional genera within the thraustochytrids are distinguishable based on the presence of
certain morphological features, except for the genus Thraustochytrium Sparrow, 1936, which serves
as a “catch-all” for the group and contains members that do not show the distinguishing characters
of any other genus.

The other traditional genera include:
 (1) Japanochytrium (Kobayashi and Ookubo, 1953), whose species are distinguishable by the
presence of a swelling (the supsoral apophysis) just below the sporulating structure,
 (2a) Schizochytrium (sensu stricto) (Goldstein and Belsky, 1964), whose members are
characterized by the sporangia undergoing vegetative mitosis (successive bipartitioning)
before the formation of spores,
 (3) Ulkenia (Gaertner, 1977), whose members are identified by the presence of an amoeboid
protoplasm being released from the sporangium prior to cleavage into zoospores,
 (4) Aplanochytrium (Bahnweg and Sparrow, 1972) emend. Leander and Porter, 2000), the
members of which display a unique gliding motility.
(source: Yokoyama et al., 2007)

For the original taxonomic classification, see also the World Register of Marine Species
(http://www.marinespecies.org/aphia.php?p=taxdetails&id=22156).

Yokoyama and Honda (2007) conducted a taxonomic rearrangement of Schizochytrium, which
resulted in the erection of two new genera, Oblongichytrium and Aurantiochytrium and an emended
description of the genus Schizochytrium. These three genera can be distinguished by different fatty
acid and pigment profiles, in addition to 18S rDNA sequence and morphological data.

 (2b) Schizochytrium (emend. Yokoyama and Honda, 2007) members possess only beta-
carotene as a carotenoid pigment and 20% arachidonic acid. Colonies are large because of
successive bipartitioning.
5
 (5) Oblongichytrium (Yokoyama and Honda, 2007) members possess canthaxanthin, beta-
carotene, abundant n-3 docosapentaenoic acid, and little n-6 docosapentaenoic acid.
Colonies are also large due to successive bipartitioning.
 (6) Aurantiochytrium (Yokoyama and Honda, 2007) members possess astaxanthin,
phoenicoxanthin, canthaxanthin, and beta-carotene as well as arachidonic acid and
docosahexaenoic acid. Colonies are smaller, but sporangia still undergo successive
bipartitioning.

2.3 Morphological Taxonomy

RT100, the mother strain of our industrial strain for producing oils rich in omega-3 fatty acids, was
grown on solid agar media (GPY or equivalent) to evaluate gross morphological characters. Figure 1
shows its displaying successive bipartitioning.
Figure 1: RT100 on solid agar, showing successive bipartitioning (arrows)

Based on several images indicating successive bipartitioning in this strain, Dr. Honda, who initially
isolated, cultured and characterized this strain, began to suspect that this strain was a close relative
to (or a member of) the Schizochytrium, Oblongichytrium, or Aurantiochytrium genera. Moreover,
RT100 did not display the amoeboid protoplast stage indicative of Aurantiochytrium limacinum or
Ulkenia.

6