Rhizopus oligosporus and Uses Therefor

FIELD OF THE INVENTION

This invention relates to microorganisms. More particularly, this invention relates to a strain of Rhizopus oligosporus and its use in the production of fermented food products.

BACKGROUND TO THE INVENTION

Fermented products are a popular food product, especially those which serve as a source of protein. Tempe is a mould fermented product which enjoys a substantial popularity throughout SouthEast Asia, and in particular Indonesia. Traditional methods of preparing tempe almost exclusively employ fermenting cooked soybean with various species of

Rhizopus but in particular Rhizopus oligosporus (R. oligosporus). The quality of tempe is very much dependent upon the quality of the starter culture with which it is inoculated. Soybean contains significant amount of carbohydrates that can be utilized for microbial fermentation however soybean is comparatively expensive and low in dietary fibre content.

The typical process for making tempe includes soaking dried soybean in water for overnight followed by removal of the seed coat, cooking and fermentation by R oligosporus for typically at least 24 hours.

SUMMARY OF THE INVENTION

It is desirable to have alternative sources of fermented food products with improved nutritional qualities but also meet industry quality standards, consumer acceptability and provide commercial advantages (in terms of

lower manufacturing costs and time). Despite intensive efforts, development of a suitable tempe products which, in part, meet this criteria has remained elusive.

Accordingly, the present inventors have responded to this long-felt need by development of a novel R. oligosporus strain which is capable of producing fermented food products such as tempe, and in particular lupin- based tempe, with shorter process and preparation times yet results in a tempe product which is equal in quality to traditional tempe products.

In a first aspect, the invention provides an isolated strain of a Rhizopus oligosporus deposited at the National Measurement Institute under Accession Number V07/031112.

In a preferred embodiment, the isolated strain of R. oligosporus V07/031112 is a biologically pure culture.

In a second aspect, the invention provides an isolated strain which is a functional counterpart of a Rhizopus oligosporus deposited at the National Measurement Institute under Accession Number V07/031112.

In a third aspect, the invention provides a composition comprising the isolated strain of R. oligosporus of the first aspect or the isolated strain of R. oligosporus of the second aspect. In certain preferred embodiments, the composition is a starting material for the manufacture of a food product for consumption.

In other preferred embodiments, the composition is a food composition which in a form of a final product ready for consumption. According to embodiments that contemplate a food composition, the food

composition further comprises at least one food ingredient.

Preferably, the composition is selected from tempe and kecap. More preferably, the composition is tempe.

In another preferred embodiment, the composition is an inoculum for use in fermentation. According to these embodiments, the inoculum further comprises a suitable carrier or diluent.

In a fourth aspect, the invention provides a method of producing a fermented food said method including the step of inoculating a substrate with the isolated strain of R. oligosporus of the first aspect or the isolated strain of R. oligosporus of the second aspect or the composition of the third aspect, to thereby produce a fermented food.

Preferably, the substrate comprises a grain legume.

More preferably, the grain legume is selected from the group consisting of a pea, a lentil, a chickpea, a lupin and a soybean. In preferred embodiments, the substrate comprises a lupin selected from the group consisting of whole lupin and dehulled lupin.

Preferably, the substrate comprises at least 10% w/w lupin.

Preferably, the fermented food is selected from tempe and kecap.

More preferably, the fermented food is tempe. In preferred embodiments of the fourth aspect, the method further includes the steps of: soaking the grain legume for between about 3 hours to 12 hours; and boiling or steaming the grain legume for between about 5 minutes and 20 minutes.

In a fifth aspect, the invention provides a fermented food produced according to the method of the fourth aspect.

Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

BRIEF DESCRIPTION OF THE DRAWINGS In order that the invention may be readily understood and put into practical effect, preferred embodiments will now be described by way of example with reference to the accompanying figures wherein like reference numerals refer to like parts and wherein:

FIG 1 Effects of lupin inclusion on the quality of lupin tempe. Mycelial growth of tempe samples was inversely related to the percentage of lupin inclusion (Table 6). This could be attributed to the fact that lupin lacks fermentable carbohydrates;

FIG 2 Tempe products after incubation for 48 hours. 1 is 100% lupin,

2 is 60:40 lupin:soy (w/w) mix with 4% tapioca, 3 is 60:40 lupin:soy (w/w) with 2% tapioca and 4 is 100% Soy; and

FIG 3 Sensory scores for overall acceptability of tempe samples. DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the isolation and purification of a strain of the fungus Rhizopus oligosporus (hereinafter referred to as R. oligosporus UWS1) which displays improved performance during production of fermented compositions and in particular, food

compositions. More particularly, R. oligosporus UWS 1 has superior performance during fermentation for the production of tempe.

R. oligosporus UWS 1 strain of the present invention has been deposited according the provisions of the Budapest Treaty at the National Measurement Institute (NMI), Melbourne, Australia on 26 November 2007 under Accession Number V07/031112.

A characteristic of R. oligosporus UWS 1 is a demonstrable improvement in the preparation of tempe in the form of decreased soaking and cooking times. Moreover, R. oligosporus UWS1 is particularly amenable to production of tempe from non-traditional sources, and in particular lupin, although without limitation thereto. The selection of R. oligosporus UWS1 was based upon an ability to ferment lupin into a consumer-grade tempe.

This is unexpected considering that lupins have low levels of carbohydrates, which serve as a substrate for microbial fermentation. Traditionally, commercially available inoculums are considered not suitable for fermentation of lupin into a consumer-grade tempe.

The present inventors have shown that the quality of lupin tempe produced using R oligosporus UWS1 is similar to that of traditional soybean tempe products as judged by consumer acceptability studies. More particularly, the lupin tempe produced according to the present invention satisfies Indonesian quality standards for tempe.

In light of the foregoing, it will be appreciated that in a general aspect the invention resides in an isolated strain of R. oligosporus Accession Number V07/031112.

In preferred embodiments, R. oligosporus Accession Number V07/031112 is a biologically pure culture.

For the purposes of this invention, by "isolated' is meant material that has been removed from its natural state or otherwise been subjected to human manipulation. Isolated material may be substantially or essentially free from components that normally accompany it in its natural state, or may be manipulated so as to be in an artificial state together with components that normally accompany it in its natural state. Isolated material may be in native, chemical synthetic or recombinant form. In the context of the present invention, by "biologically pure culture"\s meant a culture which has been purified to the extent that said culture essentially comprises one type of micro-organism. A person skilled in the art will appreciate that a "biologically pure culture" may include a minor proportion of one or more micro-organisms which are not R oligosporus UWS 1. However the levels of said minor components is such that they will never exceed the growth of the micro-organism of interest. It is envisaged that a biologically pure culture comprises at least 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% R oligosporus UWS1.

The present invention also contemplates functional counterparts of R. oligosporus UWS 1 which share a relationship with R. oligosporus UWS 1 based on common identifying characteristics. By "identifying characteristics" is meant morphological, biochemical, physiological, genetic and functional characteristics which are similar with or similar to R oligosporus UWS1. In one preferred embodiment, a functional counterpart may display a

comparable ability to ferment lupin into tempe, and in particular fermentation of tempe that meets consumer acceptability standards. A functional counterpart is inclusive of naturally occurring variants.

In another broad aspect, the invention is directed to compositions comprising R. ollgosporus UWS1. It will be appreciated that the compositions of the invention will be in a form which makes the composition suitable for a particular application such as, but not limited to, fermentation. By way of example only, the composition includes a liquid form, a solid form, a lyophilised form, or a frost form. It is also envisaged that the composition may be a granule, powder or a tablet although without limitation thereto.

In preferred embodiments, the invention is directed to food compositions and in particular, fermented food compositions comprising R. oligosporus UWS1. As hereinbefore described, the invention is particularly suited to the fermented food product tempe. In certain preferred embodiments, the invention contemplates a composition comprising R. oligosporus UWS 1 which is in a form suitable as a starting material for the manufacture of a final product for consumption, and in particular a food composition. By way of example, according to these embodiments, a starting material composition could comprise all the raw ingredients prior to undergoing fermentation to a fermented food.

In other embodiments, compositions of the invention include a food composition which is preferably in a form of a final product ready for consumption. In particular forms of these embodiments, the starting material composition has undergone fermentation to produce a food composition that

is ready for consumption.

It is envisaged in addition to R. oligosporus UWS1 , the food compositions of the present invention further comprises at least one food ingredient. It is envisaged that in embodiments which contemplate tempe, the at least one food ingredient may be selected from the group consisting of a mild acidulent (such as acetic acid), starch, sugar, spices, a flavouring and a food preservative.

In other preferred embodiments, the composition of the invention is an inoculum for fermentation. In particularly preferred embodiments, the inoculum of the present invention is amenable for fermentation of tempe using non-traditional substrates. It will be appreciated that in particular embodiments, the inoculum for R. oligosporus UWS1 may be in the form of spores that begin the growth process upon favourable growth conditions. Once activated and germinated, the resulting germ tube will grow into hyphae and, eventually, into a network of mycelia. In the case of tempe, the mycelia form a mat that binds the substrate together.

According to those embodiments which contemplate an inoculum, the composition will further comprises a suitable carrier or diluent. It will be appreciated by a person of skill in the art that the carrier or diluent should be suitable to deliver the R. oligosporus UWS 1 , and in particular the spores, as an inoculum composition. More preferably, the carrier or diluent may act as an inert support for the growth of the spores and is suitable as an addition to a food product. Non-limiting examples of a suitable carrier for R. oligosporus UWS1 include rice, wheat flour/starch, corn starch, tapioca are amenable as

carrier.

In other general aspects, the invention provides methods for the production of a fermented food such as, but not limited to, tempe and kecap. More particularly, the method of the invention includes the step of fermenting a substrate with an isolated strain of R oligosporus UWS1. Preferably, the fermented food product is tempe. In preferred embodiments, the methods of the present invention contemplate use of a substrate which is a grain legume. A person skilled in the art will appreciate that grain legumes are plants used as food in the form of unripe pods, immature seeds or mature dry seeds such as, but not limited to, peas, lentils, chickpeas, soybeans, faba beans and lupins, or a combination thereof. Preferably, the substrate comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to 100% of grain legume. The present invention is particularly suited to the production of fermentation of tempe from lupin. Lupin is one of the richest sources of plant protein, although the protein content and amino acid profile is known to vary between species. The protein content of the whole seed (32-36%) is higher than most of other pulses. Lupin has little or no starch, but is rich in dietary fibre content. Nutritionally, lupin offers some advantages as an alternative to soybean. Lupin has a lower content of trypsin inhibitors, which can interfere with digestive processes.

In the embodiments that contemplate fermentation using a substrate comprising lupin, the substrate comprises at least 10%, 15%, 20%, 25%,

30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to 100% lupin.

It will be appreciated that in those embodiments that contemplate less than 100% of a particular type of grain legume, the remainder of the substrate may comprise another grain legume such as soybean, although without limitation thereto.

For example in embodiments which contemplate less than 100% of lupin, the remainder of the substrate may comprise soybean.

The methods of the present invention contemplates use of whole grain legumes or alternatively, dehulled grain legumes. The invention is particularly suited to use of a whole lupin or a dehulled lupin kernel but without limitation thereto.

In preferred embodiments that relate to tempe, the method of production includes the steps of: (a) (optionally) heating a grain legume. In preferred embodiments, heating is by boiling for between about 5 minutes and 20 minutes. Preferably, the boiling time is between about 10 minutes and 20 minutes. More preferably, the boiling time is about 15 minutes;

(b) soaking the grain legume for between about 3 hours to 12 hours. Preferably, the soaking time is between about 5 hours and 8 hours.

More preferably, the soaking time is about 6 hours;

(c) (optionally) dehulling the grain legume in order to permit accessibility of the mycelium;

(d) boiling or steaming to soften and sterilise the grain legume. In

embodiments that contemplate steaming, the steaming time is between about 5 minutes and 20 minutes. Preferably, the steaming time is between about 10 minutes and 17 minutes. More preferably, the steaming time is about 15 minutes; and (e) fermenting the grain legume to produce tempe, which includes inoculating the grain legume with R. oligosporus UWS1 followed by incubating at about between 22°C to 35°C for between about 12-80 hours.

In preferred embodiments wherein the grain legume is lupin, fermentation with R. oligosporus UWS1 proceeds at about between 25 ⁰ C to 3O ⁰ C for between about 24 to 48 hours. In more preferred embodiments, fermentation with R. oligosporus UWS 1 proceeds by incubation at about 30°C for about 24 hours followed by incubation at 25°C for about 24 hours. In other preferred embodiments, fermentation with R. oligosporus UWS1 proceeds by incubation at about 30 ⁰ C for about 24 hours followed by incubation at 25 ⁰ C for about 48 hours.

In light of the foregoing, a person skilled in the art will appreciate that the methods for production of fermented food products of the present invention, and in particular tempe, provide advantages in terms of decreased processing times without a loss in quality of the end product. It is also envisaged that the methods of the invention utilise fermentation aids such as, but not limited to, tapioca, sugar, wheat flour/starch, corn starch, rice flour in order to assist with fermentation.

So that the invention may be readily understood and put into practical effect, the following non-limiting Examples are provided.

EXAMPLES Example 1

Development of an Inoculum and Process for Preparation of Lupin Based Tempe

Collection of traditional inoculums/cultures and the isolation of the microorganisms

A range of traditional tempe inoculums and wild species were collected from Malang, Solo, Yogjakarta, Wonogiri, Wonosari and Bandung cities of Indonesia. These cities have relatively high tempe production and consumption. Samples were coded based on the source. Tempe inoculums were sliced in to small pieces and put in 50 ml (1 :10) sterile 0.1% Tween 80. The flasks were shaken at room temperature at 120 rpm for 1 hour to get a spore suspension. The spore suspension was further diluted in a decimal dilution series (10 ^"6 , 10 ^"7 , 10 ^"8 ). One ml diluted sample of spore suspension was put in a sterile petri dish and about 10 ml of potato dextrose agar (PDA) medium (sterilised at 121 ⁰ C for 15 min and cooled to 50 ⁰ C) was added and mixed with the samples. The petri dishes were incubated at 30°C for 2-3 days. After incubation, single colonies of micro-organisms were removed to PDA slants and incubation was continued until 3 - 7 days. These steps were repeated three times to isolate pure cultures.

A total of 17 pure fungi samples were obtained. Out of which 12 were identified as Rhizopus spp. and 5 as Mucor spp. The 12 Rhizopus species were used to prepare the lupin tempe inoculums. The amylolytic and

proteolytic activities of the microorganisms were determined for the screening purpose. Based on the amylolytic and proteolytic activities and growth characteristics, 6 strains were selected for further studies. The 6 selected isolates were identified as Rhizopus oligosporus by using a range of tests and morphological characteristics. Starter and Inoculum Production

The 6 selected isolates (Table 1) were used for further evaluation as starter for tempe production. The activities of these organisms were tested by TPC, mould growth, sporulation time and tempe production. Mould growth and sporulation time of the inoculum were monitored for two days. Mould growth rates varied among different inoculums. The inoculums UWS1 and TG1 showed the best growth after 24 hours (Table 1).

The ability of the microorganism to ferment lupin was evaluated by using lupin as a substrate. Changes during the fermentation, growth of hyphae, texture and flavour of lupin tempe were monitored for three days (Table 2). The shelf life of the tempe was determined by incubating at 30 ⁰ C for one day followed by storage at room temperature. Physical changes such as growth of hypha, texture and flavour of tempe lupin were monitored at the second and third day (Table 2). The data given in Table 2 indicates that only 3 of the inoculums (TK2,

UMC1 and UWS1) produced tempe with compact texture that lasted for 3 days.

Based on the studies on the growth characteristics, tempe quality and shelf life R. oligosporus UWS 1 was selected as the best microorganism to

produce lupin based tempe. Further studies were carried out on the selected

R. oligosporus UWS1 to confirm its ability to produce a good quality lupin tempe.

Trials on reducing the process time for tempe The objective of this study was to reduce processing time for preparing lupin tempe. Different combinations of boiling, soaking and steaming times were applied (Table 3).

A dose of 0.2% of the selected inoculum UWS 1 was added to each sample and incubated at 30 ⁰ C for 24 hours followed by incubation at 25°C for 72 hours. Temperature, moisture content, pH, ammonia formation, firmness and rate of mycelium growth in lupin tempe samples were analysed to determinate the quality of lupin tempe.

The results presented in Table 4 revealed that the tempe samples prepared from lupin had higher moisture contents, lower firmness and lower mycelium growth rates than the soybean tempe. However, the lupin based samples prepared according to the new method (15 min boiling, 6 h soaking and 15 min steaming) had almost same characteristics that of prepared using the standard method (30 min boiling, overnight soaking and 30 min steaming). To improve the quality of the product prepared by the new method, further studies were carried out on the use of different fermentation aids to promote the mycelium growth rate.

Example 2

Confirmatory studies on the efficiency of the new process and the inoculum developed in our research studies

Preparation of the Inoculum (UWS 1)

White rice was mixed with water (ratio 1 :1) acidified with 0.1 N HCI up to pH 6.0. The mixture was put in a pan and cooked until the water was absorbed by the rice grains. The partially cooked rice was then steamed in a rice steamer for 30 minutes. The cooked rice (substrate) was cooled to room temperature and spread on a tray. Inoculation was made by spreading 0.5% of starter (R. oligosporus UWS1) on the substrate. The trays containing the inoculated substrates were incubated at 28°C - 32°C for 3 days. At the end of the incubation, the fresh inoculum was cut and dried in a dryer. The dried inoculum was ground into powder and stored in a refrigerator for use in tempe preparation.

Soybean Tempe (standard method)

Soybeans (100 g) were washed in cold tap water and then boiled for

30 minutes. The beans were soaked over night in water containing acetic acid ). The hulls were removed and the dehulled kernels were cleaned and then steamed for 30 minutes. The cooked kernels were cooled to about 37 ⁰ C to allow surface drying. The kernels were inoculated and filled into perforated clear plastic bags. The bags were incubated at 30 ⁰ C for 24 hours and then transferred to a control room set at 25 ⁰ C until 72 hours. The bags were sampled at 48 and 72 hours. Lupin Tempe (new method)

Dehulled lupin kernels (100 g) were washed in cold tap water and boiled for 15 minutes. The beans were soaked for 6 h in water containing acetic acid. The kernels were cleaned and then steamed for 15 minutes. The

cooked kernels were cooled to about 37°C to allow surface drying. The kernels were inoculated and filled into perforated clear plastic bags. The bags were incubated at 3O ⁰ C for 24 hours and then transferred to a control room set at 25°C until 72 hours of fermentation. The bags were sampled at 48 and 72 hours.

Effect of lupin addition and fermentation aid on the quality of tempe

By using the new inoculum and new method of using dehulled lupin beans with 15 min boiling, 6 hours soaking, and 15 min, tempe samples were prepared by sequentially replacing soybean with lupin. A range of quality parameters were studied to assess the effect of lupin incorporation. The results (Table 5, Fig 1) showed that the moisture content of tempe samples were increased by increasing the lupin level. In contrast, the firmness of tempe samples were decreased by increasing the lupin substitution (Fig 1). The maximum moisture content of soybean tempe in Indonesian Standard Industry (SII 0271-80) is 65%. The tempe sample with lupin and soybean ratio of 60:40 had the moisture content < 65% and firmness of 3.53 (kg).

To improve the mycelia growth in the tempe, studies on the application of different fermentation aids in lupin tempe preparation were carried out. Fermentation aids were added at different concentrations and combinations together with the new inoculum (R. oligisorus UWS1). The addition of 2 - 4% tapioca and 0.5% sugar as fermentation aids increased the rate of mycelium growth equal to the control (soybean) in lupin tempe during fermentation (Table 6).

The inclusion of 40% soybean and 2 - 4% tapioca improved the

quality of lupin tempe as compared to 100% lupin tempe. Lupin tempe prepared without addition of soybean or tapioca had the highest moisture content and the lowest firmness. Mycelia growth on the 100% lupin tempe was slower than lupin - soy tempe with or without the addition of tapioca. Figure 2 shows some of the tempe samples prepared. Sensory evaluation

Based on the previous results 5 samples were selected for sensory evaluation. The different lupin, soy and tapioca combinations selected for the sensory evaluation trial. Sensory evaluation of the lupin - soy tempe was carried out using a panel of 80 untrained Indonesian panellists. The samples for evaluation were sliced and steamed for 5-7 minutes. The steamed slices of tempe were immersed in a batter of rice powder containing some spices (garlic, coriander, salt, MSG) and fried at 160 - 170 ⁰ C for 3 minutes. The preparation of tempe for consumption by the above mentioned method is known as tempe keripik.

The attributes evaluated were appearance, taste, texture and overall acceptability. Nine point hedonic scale test with 1 - Dislike extremely to 9 - Like extremely were used for the study. The results for the different attributes are shown in Table 7. The highest score for appearance was for the 60:40 (lupin:soy) sample with 4% tapioca. Similar scores for taste were recorded for all samples with 60:40 lupin.soy mix with 4% tapioca. This reflected that there was no detectable negative effect of tapioca addition on the taste of lupin based tempe samples. Regarding texture, all samples recorded almost 6.5 or

above points out of 9.

The 60:40 (lupirrsoy) sample with 2 and 4% tapioca received the overall acceptability scores equal to 100% soybean sample (Fig 3).

Throughout the specification the aim has been to describe the preferred embodiments of the invention without limiting the invention to any one embodiment or specific collection of features. It will therefore be appreciated by those of skill in the art that, in light of the instant disclosure, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

TABLES

Table 1 Microbiological assay of the inoculums

Table 2 Physical changes occurring on lupin tempe fermentation at room temperature

Note: Texture : Compact = Good texture; soft and wet = Poor texture

Table 3 Trial of lupin tempe preparation with difference in processing time

Table 4 Changes in the physicochemical and microbial characteristics during lupin tempe fermentation

Table 5 Analysis data of lupin-soybean tempe after 72 hour incubation

Table 6 The effects of fermentation aids on lupin tempe

Table 7 Sensory evaluation of lupin soy tempe samples

BUDAPEST TREATY ONTHE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FORTHE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

Pro RECEIPT DSf THE CASE OF AN ORIGINAL DEPOSIT

Food Science and Technology issued pursuant to Rule 7.1 by the School of Public Health INTERNATIONAL DEPOSITARY AUTHORITY Curtin University of Technology identified at the bottom of this page BentIey WA 6102

ATTENTION:

Assoc. Prof. Vijay Jayasena

was received by it

on Treaty was received by it on

1 Where Rule 6.4(d) applies, such date is the date on which the status of International Depositary Authority was acquired ^,

FormBP/4 (sole page)

Appendix 3 page 21

BUDAPEST TREATY ON THE INTERNATIONAL

RECOGNITION OF THE DEPOSIT OF MICROORGANISMS

FORTHE PURPOSES OF PATENT PROCEDURE

INTERNATIONAL FORM

ϊTO:

Assoc. Prof. Vijay Jayasena VIABILITY STATEMENT Food Science and Technology issued pursuant to Rule 10.2 by the School of Public Health INTERNATIONAL DEPOSITARY AUTHORITY Curtin University of Technology identified on the following page Bentfey WA 6102

NAME AND ADDRESS OPTHI: PAR'Iυ TO L WHOM THE VIABILITY STATRMENT IS ISSUED

I DEPOSITOR II IDENTIFICATION OF THE MICROORGANISM

Name: Accession number given by the

Assoc. Prof. Vijay Jayasena INTERNATIONAL DEPOSITARY AUTHORITY:

Address: V07/031112

Food Science and Technology Scliool of Public Health - Date of the deposit ^" ot of the transfer : Curtin University of Technology Beαtley WA 6102 26/11/2007

III VIABILITY STATEMENT

The viability of the microorganism identified under II above was tested on : 29/01/2009. On that date, the said microorganism was

E viable

O no longer viable

1. Indicate the date of the original deposit or, where a new deposit or a transfer has been made, the most recent relevant date

(date of the new deposit or date of the transfer).

2. In the cases referred to in Rule 10.2(a) (ii) and (iii), refer to the most recent viability test.

3. Mark will) a cross the applicable box.

Fonu BP/9 (first page)

Appendix 3 page 22 to represent the

Form BP/9 (second and last page)