Federal Register of Legislation - Australian Government

Primary content

Food Standards as made
This instrument amends food standards in the Australia New Zealand Food Standards Code.
Administered by: Health
Exempt from sunsetting by the Legislative Instruments Act 2003 s 54(1), Food Standards Australia New Zealand Act 1991 s 82(2)
Registered 28 May 2009
Tabling HistoryDate
Tabled HR01-Jun-2009
Tabled Senate15-Jun-2009
Date of repeal 19 Mar 2014
Repealed by Health (Spent and Redundant Instruments) Repeal Regulation 2014
Table of contents.

 

APPLICATION A1011

Cellulase from Penicillium funiculosum as a processing aid (Enzyme)

EXPLANATORY STATEMENT

 

 

Executive Summary

 

Purpose

 

Food Standards Australia New Zealand (FSANZ) received an Application from Danisco A/S on 9 July 2008.  The Application seeks to amend Standard 1.3.3 – Processing Aids, of the Australia New Zealand Food Standards Code (the Code), to approve a cellulase enzyme preparation (EC number 3.2.1.4), produced by fermentation from a non-toxigenic and non-pathogenic strain of Penicillium funiculosum as a processing aid. 

 

Processing aids are required to undergo a pre-market safety assessment before approval for use in Australia and New Zealand.  Cellulases sourced from Aspergillus niger, Trichoderma reesei and T. viride are currently listed as permitted processing aids in Standard 1.3.3 – Processing aids in the Table to clause 17 – Permitted enzymes of microbial origin.

 

It is proposed that this cellulase enzyme preparation will be used in the brewing and distilling industries to allow for more efficient processing and improved product quality whilst also reducing the overall costs of production. 

 

The enzyme preparation meets international specifications for enzymes.  The Joint FAO/WHO Expert Committee on Food Additives (JECFA) has evaluated and published specifications for the cellulase preparation from Penicillium funiculosum.  The Applicant has stated that cellulase from P. funiculosum is affirmed as Generally Recognized As Safe (GRAS) (this is a self-affirmation) in the United States and Brazil.  It has also been evaluated and accepted for use in Russia and is currently under evaluation in Denmark.  This enzyme preparation is currently used in the brewing and distilling industries in Belgium, Germany, Finland, The United Kingdom, Nigeria, Poland, Slovenia, Thailand and Ukraine.

 

The Application was assessed under the General Procedure.

 

Safety Assessment

 

On the basis of the following considerations, it was concluded that there are no public health and safety concerns associated with the proposed use of cellulase derived from Penicillium funiculosum:

 


·                cellulase has very low acute and repeat-dose toxicity in rats, and is not genotoxic in vitro;

 

·                there is a history of safe use of the source organism and cellulase in the food industry;

 

·                people are already exposed to both the source organism and cellulase given their natural occurrence in food;

 

·                cellulase derived from a number of other microbial sources is already approved for the same use in Australia and New Zealand; and

 

·                cellulase derived from the same source is an approved processing aid in the USA, Brazil and certain parts of Europe.

 

The enzyme is to be used as a processing aid only and any enzyme residues present in the final food product are unlikely to be active and would be metabolised in the gastrointestinal tract in a similar manner to any other ingested protein.

 

Labelling

 

The enzyme preparation is produced from a non-genetically modified, classical source organism, P. funiculosum.  The enzyme cellulase is not considered to be allergenic and there are no potential allergens involved in the manufacturing process.  Therefore, there would be no labelling requirement under Standard 1.2.3 for potential allergens.

 

Assessing the Application

 

In assessing the Application and the subsequent development of a food regulatory measure, FSANZ has had regard to the following matters as prescribed in section 29 of the Food Standards Australia New Zealand Act 1991 (FSANZ Act):

 

·                whether costs that would arise from an amendment to the Code to permit the use of the enzyme preparation, cellulase, produced by fermentation from a non-toxigenic strain of P. funiculosum would outweigh the direct and indirect benefits to the community, Government or industry; 

 

·                there are no other measures that would be more cost-effective than a variation to Standard 1.3.3 that could achieve the same end;

 

·              there are no relevant New Zealand standards; and

 

·                there are no other relevant matters.

 

Decision

 

FSANZ approves the draft variation to the Table to clause 17 of Standard 1.3.3 – Processing Aids, to permit the use of the enzyme cellulase produced from a non-toxigenic strain of Penicillium funiculosum as a processing aid. 

 

Reasons for Decision

 

An amendment to the Code approving the use of the cellulase enzyme preparation as a processing aid in Australia and New Zealand is approved on the basis of the available scientific evidence for the following reasons:


·                A detailed Safety Assessment Report has concluded that the use of enzyme does not raise any public health and safety concerns.

 

·                The source organism is a classical non-genetically modified strain of P. funiculosum and is regarded as non-pathogenic and non-toxigenic.

 

·                Use of the enzyme sourced from P. funiculosum is expected to provide technological benefit to food manufacturers.

 

·                The regulation impact assessment has concluded that the benefits of permitting the enzyme as a processing aid outweigh any costs associated with its use.

 

·                There are no other measures that would be more cost-effective than a variation to Standard 1.3.3 that could achieve the same end.

 

·                The proposed draft variation to the Code is consistent with the section 18 objectives of the FSANZ Act.

 

·                There are no New Zealand standards relevant to the decision.

 

Consultation

 

Public submissions were invited on the Assessment Report.  Comments were specifically requested on the scientific aspects of the Application, in particular, information relevant to the safety assessment of the enzyme cellulase sourced from P. funiculosum.

 

A total of three submissions were received.  A summary of these is provided in Attachment 2 to this Report.

 

As this Application was assessed under General Procedure, there was only one round of public comment.  FSANZ gave careful consideration to the submissions received in its decision.  The main issues raised in public comments are discussed in this Report.  Neither the preferred approach, nor the draft variation to the Code, has altered from that proposed in the Assessment Report. 

 


CONTENTS

Introduction.. 2

1.     The Issue / Problem.. 2

2.     Current Standard. 2

2.1       Historical background. 2

2.2       Current Standard. 3

2.3       International Regulatory Standards. 3

2.4       Nature of the Enzyme and Source of Organism.. 4

2.5       Technological purpose of the enzyme. 4

2.6       Labelling issues. 4

3.     Objectives. 4

4.     Questions to be answered. 5

RISK ASSESSMENT.. 5

5.     Risk Assessment Summary. 5

5.1       Safety Assessment 5

5.2       Dietary Exposure Assessment of the cellulase enzyme preparation. 6

5.3       Technological Justification. 6

5.4       Production of the enzyme. 6

5.5       Allergenicity. 7

Risk Management.. 7

6.     Issues raised. 7

6.1       Risk Management Strategy. 7

7.     Options. 7

8.     Impact Analysis (RIS ID:  9852) 7

8.1       Affected Parties. 8

8.2       Benefit Cost Analysis. 8

8.3       Comparison of Options. 8

Communication and Consultation Strategy.. 9

9.     Communication. 9

10.       Consultation. 9

10.1     Issues Raised in Public Consultation. 9

10.2     World Trade Organization (WTO) 10

Conclusion.. 10

11        Conclusion and Decision. 10

11.1     Reasons for Decision. 10

12.       Implementation and Review.. 11

Attachment 1 - Draft variation to the Australia New Zealand Food Standards Code. 12

Attachment 2 - Summary of Issues Raised in Public Submissions. 13

Attachment 3 - Safety Assessment Report. 14

Attachment 4 - Food Technology Report. 21

 


Introduction

 

Food Standards Australia New Zealand (FSANZ) received an Application from Danisco A/S on 9 July 2008.  The Application seeks to amend Standard 1.3.3 – Processing Aids, of the Australia New Zealand Food Standards Code (the Code), to permit a cellulase enzyme preparation (EC number 3.2.1.4), produced by fermentation from a non-toxigenic and non-pathogenic classical strain of Penicillium funiculosum as a processing aid. 

 

This cellulase enzyme preparation is proposed to be used in the brewing and distilling industries for its potential to allow for more efficient processing and improved product quality whilst also reducing the overall costs of production.  

 

In brewing, cellulase is used during the mashing stage to reduce the viscosity of the wort and to improve the separation of the wort from the spent grains.  In distilling, cellulase is used to reduce the viscosity of the mash and to improve the overall efficiency of manufacture.

 

1.        The Issue / Problem

 

The Applicant proposes the use of cellulase derived from P. funiculosum as a processing aid.  A processing aid is a substance used in the processing of raw materials, foods or ingredients, to fulfil a technological purpose relating to treatment or processing, but does not perform a technological function in the final food. 

 

A processing aid used in the course of manufacture of a food must be used at the lowest level necessary to achieve a function in the processing of that food, irrespective of any maximum permitted level specified.  Processing aids are prohibited from use in food in Australia and New Zealand unless there is a specific permission for them in Standard 1.3.3.  Processing aids (which includes enzymes) are required to undergo a pre-market assessment before they are approved for use in food manufacture in Australia and New Zealand. 

 

An assessment (which includes a safety assessment) of the use of cellulase derived from P. funiculosum is required before an approval for its use can be given (i.e. listed in Standard 1.3.3).

 

2.        Current Standard

 

2.1         Historical background

 

Cellulases and related enzymes have been found to have great potential in various biotechnology arenas and are used in the food, brewery, distilling and wine industries as well as in animal feed, agriculture, paper and pulp industries, textile and laundry and in other related applications[1]


2.2         Current Standard

 

Standard 1.3.3 regulates the use of processing aids in food manufacturing.  The Table to clause 17 – Permitted enzymes of microbial origin contains a list of permitted enzymes of microbial origin for use as processing aids. 

 

Clause 1 of Standard 1.3.3 defines a processing aid as:

 

Processing aid means a substance listed in clauses 3 to 18, where –

 

(a)          the substance is used in the processing of raw materials, foods or ingredients, to fulfil a technological purpose relating to treatment or processing, but does not perform a technological function in the final food; and

(b)          the substance is used in the course of manufacture of a food at the lowest level necessary to achieve a function in the processing of that food, irrespective of any maximum permitted level specified.

 

Cellulase sourced from Aspergillus niger, Trichoderma reesei and T. viride are currently listed as permitted processing aids in Standard 1.3.3 in the Table to clause 17.

 

Cellulase from the microbial source organism, P. funiculosum, is not currently listed in the Table to clause 17 or any other table in Standard 1.3.3.

 

2.3         International Regulatory Standards

 

Commercial cellulase enzyme preparation sourced from P. funiculosum complies with internationally recognised specifications for the production of enzymes, specifically the specifications of the Joint FAO/WHO Expert Committee on Food Additives (JECFA)[2] and Food Chemicals Codex[3] specifications for enzyme preparations.  Detailed specifications for cellulase from P. funiculosum have also been revised and published at the 55th JECFA meeting (2000) superseding tentative specifications prepared at the 31st JECFA meeting (1987).  No Acceptable Daily Intake (ADI) has been allocated to this enzyme preparation.

 

The Applicant has stated that cellulase from P. funiculosum is affirmed as Generally Recognized As Safe (GRAS) (this is a self-affirmation) in the United States and Brazil.  Cellulase from P. funiculosum is currently sold and used as a processing aid for the brewing, wine and ethanol industries in the United States, Brazil, Belgium, Germany, Finland, the United Kingdom, Nigeria, Poland, Slovenia, Thailand and Ukraine.  This enzyme has been evaluated and accepted in Russia where each new enzyme needs to be approved and listed as permitted, and has been positively listed for use in foods in the Ukraine.   

 


Within Europe, food enzymes are considered processing aids and are excluded from the Food Additive Framework Directive.  France, Denmark and the UK have legislation covering all food-use enzymes.  The Applicant stated that this enzyme preparation is currently under evaluation in Denmark.  In other EU countries food enzymes should be proven safe before used in food production according to the General EU law. 

 

2.4         Nature of the Enzyme and Source of Organism

 

The cellulase enzyme preparation sourced from P. funiculosum is a mixture of a number of cellulose degrading enzymes including three endoglucanases, two cellobiohydrolases and a β-glucosidase.  These enzymes work by catalysing the cellulolysis (or hydrolysis) of cellulose.  Substrates for cellulase are the 1,4-b-D-glucosidic linkages in cellulose, lichenin and cereal b-D-glucans, and the 1,4-linkages in b-D-glucans that also contain 1,3-linkages. The mixture also contains ‘minor enzyme activities’ attributed to b-glucanase, which hydrolyses 1,3- or 1,4-linkages of b-D-glucans; xylanase, which hydrolyses 1,4-b-D-xylosidic linkages in xylans; and glucan endo-1,3-b-D-glucosidase, which hydrolyses 1,3-b-D-glucosidic linkages in 1,3-b-D-glucans. The presence of these minor enzymes is apparently important for the functioning of the cellulase mixture.

 

The source micro-organism is claimed to be a food grade non pathogenic and non toxigenic strain of P. funiculosum developed via classical strain improvements only and is not genetically modified.

 

2.5         Technological purpose of the enzyme

 

The cellulase enzyme preparation sourced from P. funiculosum is used as a processing aid to catalyse the enzymatic hydrolysis of cellulose and thereby convert celluloses into smaller units such as glucose and other soluble sugar units.  This cellulase enzyme preparation is proposed to be used in the brewing and distilling industries for its potential to allow for more efficient processing and improved product quality whilst also reducing the overall costs of production.  In brewing cellulase is used during the mashing stage to reduce the viscosity of the wort and to improve the separation of the wort from the spent grains.  In distilling cellulase is used to reduce the viscosity of the mash and to improve the overall efficiency of manufacture.

 

2.6         Labelling issues

 

As a processing aid, the cellulase enzyme preparation would not be required to be labelled on the final food product.

 

No allergenic materials (as listed in the Table to clause 4 in Standard 1.2.3 – Mandatory warning and advisory statements and declarations) are present in the manufacturing process or in the final cellulase enzyme preparation; consequently there would be no labelling requirement necessitated due to any requirements within Standard 1.2.3.

 

3.        Objectives

 

In developing or varying a food standard, FSANZ is required by its legislation to meet three primary objectives which are set out in section 18 of the FSANZ Act.  These are:

 

·                the protection of public health and safety;

 


·                the provision of adequate information relating to food to enable consumers to make informed choices; and

 

·                the prevention of misleading or deceptive conduct.

 

In developing and varying standards, FSANZ must also have regard to:

 

·                the need for standards to be based on risk analysis using the best available scientific evidence;

 

·                the promotion of consistency between domestic and international food standards;

 

·                the desirability of an efficient and internationally competitive food industry;

 

·                the promotion of fair trading in food; and

 

·                any written policy guidelines formulated by the Ministerial Council[4].

 

4.        Questions to be answered

 

The key questions which FSANZ considered as part of the assessment were:

 

·                What was the risk to public health and safety from the use of cellulase derived from the microbial source organism, P. funiculosum?

 

·                Were there any risk management measures required to protect public health and safety?

 

·                Did the regulatory impact statement (RIS) conclude that the benefits of permitting use of the enzyme outweighed any costs associated with its use?

 

RISK ASSESSMENT

 

5.        Risk Assessment Summary

 

5.1         Safety Assessment

 

The aim of the safety assessment was to evaluate the safety of cellulase, derived from P. funiculosum (strain PF8/403-M), for use as a food processing aid.  On the basis of the following considerations, it was concluded that there are no public health and safety concerns associated with the proposed use of cellulase derived from this source:

 

·                cellulase has very low acute and repeat-dose toxicity in rats, and is not genotoxic in vitro;

 

·                there is a history of safe use of the source organism and cellulase in the food industry;

 


·                people are already exposed to both the source organism and cellulase given their natural occurrence in food;

 

·                cellulase derived from a number of other microbial sources is already approved for the same use in Australia and New Zealand; and

 

·                cellulase derived from the same source is an approved processing aid in the US, Europe and Brazil.

 

On the basis that cellulase is a natural part of the diet and that the small concentrations of cellulase (if any) present in the final food would be metabolised as any other dietary protein, it is unnecessary to set an ADI.  Therefore, the ADI can be considered to be ‘not specified’.

 

A full safety assessment is provided in Attachment 3.  

 

5.2         Dietary Exposure Assessment of the cellulase enzyme preparation

 

There are no nutritional or dietary implications in approval of this cellulase preparation since there will be no or very little, residual inactivated enzyme present in the final food and any residues would be metabolised like any other protein.  Extensive dietary modelling was not required for the use of the enzyme since it will be used as a processing aid and the majority of the enzyme will be removed from the final product. 

 

5.3         Technological Justification

 

The use of the cellulase enzyme preparation sourced from P. funiculosum as a processing aid is technologically justified to catalyse the enzymatic hydrolysis of cellulose and thereby convert celluloses into glucose and soluble sugars. 

 

The use of the cellulase enzyme would be advantageous in the production of beer to hydrolyse excess β-glucans and ultimately reduce the viscosity of the wort.  In the distilling industry the addition of cellulase degrades non-starch polysaccharides such as β-D-glucans and arabinoxylans, resulting in a decrease in mash viscosity, more efficient processing and a potential increase in alcohol yields.

 

A Food Technology Report on the technological function of the enzyme is provided in Attachment 4

 

5.4         Production of the enzyme

 

The cellulase enzyme preparation is produced via a submerged fermentation process utilising appropriate substrate and nutrients under appropriate manufacturing practices.  After the fermentation process is complete, the biomass is removed by centrifugation and filtration processes.  The remaining fermentation broth, containing the enzyme, is subsequently concentrated. 

 

This concentrated enzyme solution is standardised and preserved using a compound such as sodium benzoate.  Filtration is used as a final purification step for the cellulase preparation.  Throughout the manufacturing process, cellulase is monitored and carefully controlled by analytical and quality assurance procedures which ensure that the final product complies with the appropriate specifications and is of the highest quality for use as a processing aid in food manufacturing applications.

 


5.5         Allergenicity

 

No allergenic materials (as listed in Table to clause 4 in Standard 1.2.3) have been declared or are found to be present in the manufacturing process or in the final enzyme preparation.

 

The cellulase enzyme preparation itself is not considered to be allergenic.

 

Risk Management

 

6.        Issues raised

 

6.1         Risk Management Strategy

 

This Approval Report concludes that the use of cellulase sourced from P. funiculosum as a processing aid does not pose a public health and safety risk and its use is technologically justified. 

 

7.        Options

 

FSANZ is required to consider the impact of various regulatory (and non-regulatory) options on all sections of the community, especially relevant stakeholders who may be affected by this Application.  The benefits and costs associated with the proposed amendment to the Code have been analysed using regulatory impact principles.

 

Enzymes (as processing aids in the Code) used in Australia and New Zealand are required to be listed in Standard 1.3.3,

 

Two options have been identified for this Application:

 

Option 1:        Reject the Application and maintain status quo

 

Option 2:        Amend Standard 1.3.3 to permit the use of cellulase sourced from P. funiculosum as a processing aid.

 

8.        Impact Analysis (RIS ID:  9852)

 

In developing food regulatory measures for adoption in Australia and New Zealand, FSANZ is required to consider the impact of all options on all sectors of the community, including consumers, the relevant food industries and governments.  The regulatory impact assessment identifies and evaluates, though is not limited to, the costs and benefits arising from the regulation and its health, economic and social impacts.  The regulatory impact analysis is designed to assist in the process of identifying the affected parties and the likely or potential impacts the regulatory provisions will have on each affected party.  Where medium to significant competitive impacts or compliance costs are likely, FSANZ will seek further advice from the Office of Best Practice Regulation (OBPR) and estimate compliance costs of regulatory options. 

 

FSANZ has conducted, and OBPR has subsequently approved, an assessment of this Application which has concluded that there was no business compliance costs involved and/or minimal impact and consequently a Regulation Impact Statement (RIS) is not required.

 


8.1         Affected Parties

 

The affected parties to this Application include the following:

 

·                the enzyme manufacturers and suppliers

 

·                consumers of beer and distilled alcoholic beverages

 

·                food industry, including importers of food, wishing to produce and market food/beverage products manufactured using this enzyme

 

·                the Governments of Australia (Federal, State and Territory) and New Zealand

 

8.2         Benefit Cost Analysis

 

8.2.1      Option 1 – Reject the Application

 

This option is the status quo, with no changes to the Code.

 

Rejecting the Application would disadvantage consumers and relevant food industries where the enzyme could provide a technological function.

 

8.2.2      Option 2 – Permit the use of cellulase sourced from P. funiculosum as a processing aid.

 

This option does provide positive benefits to consumers and the food industry.  The brewing and distilling industries will benefit from having an alternative source of the cellulase enzyme, which improve process efficiencies and product quality whilst reducing the overall costs of production.  The use of the enzyme does not raise any public health and safety concerns.  

 

There should not be any significant compliance costs for government enforcement agencies since they would not need to analyse for the presence of the enzyme.  The use of enzymes to treat food during their manufacture does not require labelling so it would not be expected that enforcement agencies would need to analyse for the presence or otherwise of the enzyme in any final food for compliance.  There should also be no added costs to consumers.

 

8.3         Comparison of Options

 

In assessing Applications, FSANZ considers the impact of various regulatory (and non-regulatory) options on all sectors of the community, including consumers, food industries and governments in Australia.

 

For this Application, Option 1, the status quo, does not have any additional benefits or cost for food industry, consumers and/or government.

 

Hence Option 2 is favoured since there are potential benefits for the food manufacturing industry, as well as consumers.  Such benefits are most likely to include providing manufacturers with an alternative source of the enzyme.  No significant adverse costs have been identified with option 2 for government stakeholders.  Overall, the benefits outweigh any costs for Option 2.  Therefore Option 2 is the preferred option.

 


Communication and Consultation Strategy

 

9.        Communication

 

FSANZ acknowledges that this Application will be of interest to a broad range of stakeholders and has applied a general communication strategy.  This involved notifying the public of the availability of the Assessment Report for public comment and making reports available on the FSANZ website.  It is considered that this Application is a routine matter. 

 

The Applicant and individuals and organisations that make submissions on this Application will be notified at each stage of the assessment of the Application.  Once the FSANZ Board has approved the variation to the standard, that decision will be notified to the Ministerial Council.  The Applicant and stakeholders, including the public, will be notified of that notification and any gazettal of changes to the Code in the national press and on the FSANZ website.

 

FSANZ will continue to monitor media response and reaction to this Application.  FSANZ also provides an advisory service to the jurisdictions on changes to the Code.

 

10.      Consultation

 

10.1          Issues Raised in Public Consultation

 

The Assessment Report was advertised for public comment between 28 October 2008 and 9 December 2008.  Comments were specifically requested on the scientific aspects of this Application including:

 

·                safety considerations;

·                technological justification;

·                other scientific aspects; and

·                costs and benefits. 

 

As this Application was assessed under a General Procedure, there was one round of public comment. 

 

A total of 3 submissions were received with all submissions supporting the Application.  A summary of these is provided in Attachment 2 to this Report. 

 

FSANZ has taken the submitters’ comments into account in preparing this Approval Report for the Application. 

 

10.1.1         Clarification requested for an ambiguity identified in the Assessment Report for the specific countries where this enzyme has been evaluated and is accepted for use.

 

10.1.1.1      FSANZ response

 

One Submitter noted that there was an ambiguity in page i of the Assessment Report which stated ‘…has also been evaluated and accepted for use in Russia and Brazil’, and in page 3 of the Assessment Report which stated ‘…has been evaluated and accepted in Russia and the Ukraine where each new enzyme needs to be approved and listed as permitted’. 


This enzyme has been accepted as GRAS in Brazil and has been listed on the ‘positive list of food additives (including enzymes)’ in the Ukraine; it has also been evaluated and accepted for use in Russia.  The specific permissions for the use of this enzyme internationally have been clarified within this report, see section 2.3. 

 

10.2       World Trade Organization (WTO)

 

As members of the World Trade Organization (WTO), Australia and New Zealand are obligated to notify WTO member nations where proposed mandatory regulatory measures are inconsistent with any existing or imminent international standards and the proposed measure may have a significant effect on trade.

 

Amending the Code to approve cellulase as a processing aid is unlikely to have a significant effect on trade.  The enzyme preparation is consistent with the international specifications for food enzymes of JECFA and the Food Chemicals Codex so there does not appear to be a need to notify the WTO.  For these reasons FSANZ decided not to notify the WTO under either the Technical Barriers to Trade or Sanitary and Phytosanitary Measures Agreements. 

 

Conclusion

 

11       Conclusion and Decision

 

This Application has been assessed against the requirements of section 29 of the FSANZ Act.  FSANZ recommends the proposed draft variation to Standard 1.3.3.    Neither the preferred approach nor the draft variation has altered from that proposed in the Assessment Report. 

 

This Approval Report concludes that the use of the cellulase enzyme sourced from P. funiculosum as a processing aid is technologically justified and does not pose a public health and safety risk.

 

An amendment to the Code to give approval to the use of the enzyme cellulase sourced from P. funiculosum as a processing aid in Australia and New Zealand is approved on the basis of the available scientific information.  The proposed draft variation (which is the same as that proposed at Assessment) is provided in Attachment 1.  FSANZ has not made any amendments to the draft variation as a result of consideration of the submissions received.

 

Decision

 

FSANZ approves the draft variation to the Table to clause 17 of Standard 1.3.3 – Processing Aids, to permit the use of the enzyme cellulase produced from a non-toxigenic strain of Penicillium funiculosum as a processing aid.  

 

11.1       Reasons for Decision

 

An amendment to the Code approving the use of the cellulase enzyme preparation as a processing aid in Australia and New Zealand is approved on the basis of the available scientific evidence for the following reasons:

 

·                A detailed safety assessment has concluded that the use of enzyme does not raise any public health and safety concerns.

 

·                The source organism is a classically bred non-genetically modified strain of P. funiculosum and is regarded as non-pathogenic and non-toxigenic.


·                Use of the enzyme sourced from P. funiculosum is expected to provide technological benefit to manufacturers.

 

·                The regulation impact assessment has concluded that the benefits of permitting the enzyme as a processing aid outweigh any costs associated with its use.

 

·                There are no other measures that would be more cost-effective than a variation to Standard 1.3.3 that could achieve the same end.

 

·                The proposed draft variation to the Code is consistent with the section 18 objectives of the FSANZ Act.

 

·                There are no New Zealand standards relevant to the decision.

 

12.      Implementation and Review

 

The FSANZ Board’s decision will be notified to the Ministerial Council.  Following notification, the proposed draft variation to the Code is expected to come into effect on gazettal, subject to any request from the Ministerial Council for a review of FSANZ’s decision.

 

ATTACHMENTS

 

1.       Draft variation to the Australia New Zealand Food Standards Code

2.       Summary of issues raised in public submissions

3.       Safety Assessment Report

4.       Food Technology Report

 


Attachment 1

 

Draft variation to the Australia New Zealand Food Standards Code

 

Subsection 87(8) of the FSANZ Act provides that standards or variations to standards are legislative instruments, but are not subject to disallowance or sunsetting

 

To commence:            On gazettal

 

[1]          Standard 1.3.3 of the Australia New Zealand Food Standards Code is varied by inserting the following Source in Column 2 of the Table to clause 17 for the enzyme Cellulase in Column 1

 

Penicillium funiculosum

 

 


Attachment 2

 

Summary of Issues Raised in Public Submissions

 

Submitter

Option

Comments

Food Technology Association of Australia

 

2

·       Supports progression

New Zealand Food Safety Authority

2

·     Supports progression

·     Supports the conclusion that the use of the enzyme is technologically justified and that no public health or safety concerns were identified.

 

Queensland Health

2

·     Supports progression

·     Supports the conclusion that the use of the enzyme is technologically justified and does not pose a public health or safety risk.

·     Clarification requested for an ambiguity identified in the Assessment Report for the specific countries where this enzyme has been evaluated and is accepted for use 

 

 

 

 


Attachment 3

 

Safety Assessment Report

 

A1011 – Cellulase from Penicillium funiculosum as a processing aid (Enzyme)

 

Summary and Conclusions

 

The aim of the current assessment was to evaluate the safety of cellulase, derived from Penicillium funiculosum strain PF8/403-M, for use as a food processing aid.  On the basis of the following considerations, it was concluded that there are no public health and safety concerns associated with the proposed use of cellulase derived from this source:

 

·                cellulase has very low acute and repeat-dose toxicity in rats, and is not genotoxic in vitro;

·                there is a history of safe use of the source organism and cellulase in the food industry;

·                people are already exposed to both the source organism and cellulase given their natural occurrence in food;

·                cellulase derived from a number of other microbial sources is already approved for the same use in Australia and New Zealand; and

·                cellulase derived from the same source is an approved processing aid in the US, Europe and Brazil.

 

On the basis that cellulase is a natural part of the diet and that the small concentrations of cellulase (if any) present in the final food would be metabolised as any other dietary protein, it is unnecessary to set an ADI.  Therefore the ADI can be considered to be ‘not specified’.

 

Background

 

Application A1011 seeks approval for the use of the enzyme cellulase, derived from P. funiculosum strain PF8/403-M, and intended for use as a processing aid in brewing and distilling.  In both cases, cellulase is added during mashing to degrade polysaccharides and decrease the viscosity of the wort or mash. 

 

Cellulase (EC No. 3.2.1.4) derived from three microorganisms, namely Aspergillus niger, Trichoderma reesei and Trichoderma viride, is approved for use as a processing aid in Australia and New Zealand (Standard 1.3.3 of the Code).  In addition, a number of Penicillium species are already approved as the source organism for a number of other enzymes used as processing aids (e.g. P. lilacinum as a source of dextranase; P. camembertii as a source of monoacylglycerol lipase; and P. roquefortii as a source of triacylglycerol lipase). 

 

Cellulase derived from P. funiculosum is generally recognised as safe (GRAS) in the US and Brazil for use in brewing, wine making and distilling.  In addition, it is approved for food use in Russia and is sold for use in brewing in Europe.

 

Cellulase derived from P. funiculosum was evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1987 at its 31st Meeting.  No acceptable daily intake (ADI) was established because no safety data were available to the committee (WHO 1987).  JECFA specifications for cellulase derived from P. funiculosum have been established (FAO 2006a).

 

The current assessment was undertaken to determine the safety of cellulase derived from P. funiculosum (strain PF8/403-M), which is not currently approved in the Code. 


Hazard Assessment

 

Chemistry

 

Information on the physicochemical properties, product specification and impurity profile for cellulase derived from P. funiculosum strain PF8/403-M is provided in the Food Technology Report. 

 

Toxicity of cellulase

 

The Applicant submitted a number of unpublished toxicity studies on cellulase derived from P. funiculosum (designated Cellulase 2000L) including a rat acute oral toxicity study and two in vitro genotoxicity studies.  A rat subchronic feeding study on a concentrated liquid derived from P. funiculosum was also submitted.  The strain of P. funiculosum from which the test materials were derived was not specified.  These studies were quality assured and conducted according to principles of Good Laboratory Practice (GLP).  In addition, all studies complied with national and/or international test guidelines.  The toxicological database was considered adequate for characterising the hazard posed by cellulase derived from P. funiculosum strain PF8/403-M.  

 

Acute toxicity

 

McRae L (1995) Cellulase 2000L: acute oral toxicity to the rat. Lab: Huntingdon Research Centre Ltd, Cambridgeshire, England. Report No. RNP 461a/950521/AC. Sponsor: Rhone Poulec, ABM Brewing and Enzymes Group, Cheshire, England. Unpublished.

 

Guidelines/GLP: OECD Test Guideline 401 ‘Acute oral toxicity’ (Adopted 24 February 1987); Statement of compliance with OECD, US EPA, Japanese, UK and EC standards of Good Laboratory Practice (GLP); QA Statement.

 

Experimental: Groups of five male and five female Hsd/Ola:Sprague-Dawley (CD) rats (sourced from Harlan Olac Ltd, Bicester, Oxon, England; 95-124 g bodyweight; 4-7 weeks old) were given a single oral gavage dose of cellulase [sourced from Rhone Poulec, ABM Brewing and Enzymes Group, Cheshire, England; Batch No. WE 540; activity = 2041 units/g solution; specific activity = 35557 units/g total organic solids (TOS)] at a nominal dose of 2000 mg/kg bw.  The test material was given as an undiluted liquid as supplied by the sponsor.  Rats were housed 5/cage/sex under standard conditions, with food and water available ad libitum from approximately four hours post-dose.  Rats were observed twice daily for mortalities and clinical signs.  Bodyweights were recorded prior to dosing and at days 8 and 15. Survivors were sacrificed on Day 15 then necropsied. 

 

Findings: There were no deaths. Piloerection was observed in all rats within four minutes of dosing and resolved by day 2.  Bodyweight gains in all rats were normal. Necropsy revealed no macroscopic abnormalities. The LD50 was > 2000 mg/kg bw (equivalent to 4082 units of enzyme activity). 

 

Subchronic toxicity

 

Jones LJ, Bartlett AJ & Brooks PN (1998) Penicillium funiculosum concentrated liquid: Ninety day repeated dose oral (gavage) toxicity study in the rat. Lab: Safepharm Laboratories Ltd, Derby UK. SPL Project No. 854/004. Sponsor: Rhodia Ltd, Stockport, Cheshire, UK. Unpublished.

 


Guidelines/GLP: OECD Test Guideline 408 ‘Repeated dose 90-day oral toxicity study in rodents’; Statement of compliance with OECD standards of GLP; QA Statement.

 

Experimental: P. funiculosum concentrated liquid (provided by the sponsor; Batch No. TSL35/97; activity = 3823 U/g solution; specific activity = 47137 U/g TOS) was diluted in water and given to groups of 10 Crl:CD®BR rats/sex/dose (sourced from Charles River Ltd, Margate, Kent, UK; 7-8 weeks old; bodyweight range of 158-221 and 142-199 g in males and females, respectively) by oral gavage at nominal doses of 0, 50, 250 or 1000 mg/kg bw/day for 90 days.  The dose selection was based on the results of an unpublished 14-day ranging-finding study.  The dose volume was 5 mL/kg bw.  Rats were housed in groups of up to four/sex under standard conditions, with food and water available ad libitum.  

 

Observations for mortalities and clinical signs were made twice daily.  Bodyweights were recorded the day prior to the commencement of dosing and thereafter on a weekly basis.  Food consumption of each caged group was recorded weekly.  Water consumption of each caged group was recorded daily.  Ophthalmoscopic examinations were performed prior to treatment and during Week 12. Non-fasted blood was obtained from each rat on Day 90 and the standard range of haematology and clinical chemistry parameters analysed.  No urinalysis was performed.  All surviving rats were killed at the end of the study and necropsied.  The following organs were weighed: adrenals, brain, heart, kidneys, liver, ovaries, spleen and testes.  The standard range of organs/tissues were histopathologically examined in the control and high-dose groups.  Data were analysed using appropriate statistical tests.

 

Findings: There were no deaths and no treatment-related clinical signs.  Mean absolute bodyweights of treated males was lower (4-8%) than control from Day 14, with a concomitant lower bodyweight gain over the same period.  Given the relatively small magnitude of the difference, the absence of a dose-response relationship or a similar effect in females, this observation was not considered treatment-related or toxicologically-significant.  Food and water consumption, food conversion efficiency (bodyweight gain/food consumption) haematology and clinical chemistry parameters were unaffected by treatment.  There were no treatment-related macroscopic or microscopic abnormalities.  

 

Absolute organ weights were unaffected by treatment.  In high-dose females, relative brain weights were significantly higher (p<0.05) than the control (0.6809+0.0546% versus 0.6318+0.0369% of bodyweight, respectively).  However, this result was not considered toxicologically-significant as it fell within the historical control range in age and sex-matched rats of the same strain (0.4908-0.8044) as provided in the study report.  The no observed adverse effect level (NOAEL) was 1000 mg/kg bw/day, the highest dose tested, based on the absence of any toxicological effects.

 

Genotoxicity

 

The results of two in vitro genotoxicity assays with cellulase (sourced from Rhone Poulec, ABM Brewing and Enzymes Group, Cheshire, England; Batch No. WE 540; activity = 2041 units/g solution; specific activity = 35557 units/g TOS) are summarised in Table 1.  The study of Jones and Gant (1995) was in accordance with OECD Test Guideline 471 (Bacterial Reverse Mutation Test), while the study of Adams et al (1995) was in accordance with OECD Test Guideline 473 (In Vitro Mammalian Chromosome Aberration Test).  Both assays were conducted in the presence and absence of an exogenous source of metabolic activation (9000 g supernatant containing microsomal enzymes prepared from Aroclor™-induced SD rat liver).  In both studies, positive and negative controls gave expected results.

 


Table 1:  In vitro genotoxicity of cellulase

 

End point

Test system

Concentration

Result

Reference

Reverse mutation

Salmonella typhimurium strains TA98, TA100, TA1535 & TA1537

0-204.10 units/plate

Negative

Jones & Gant (1995)

[GLP; QA]

Chromosomal aberration

Human lymphocytes

0-5.86 U/mL

Negative

Adams et al (1995)

[GLP; QA]

GLP = statement of compliance with principle of GLP; QA = quality assured

 

Discussion

 

P. funiculosum is a common fungi found in soil and food, and which is not known to be pathogenic, toxic or allergenic to humans.  Further, there is a safe history of use of P. funiculosum strain PF8/403-M, which has been used to produce food-grade enzyme products since 1991 such as b-glucanase, endo-1,3(4)-beta-glucanase, xylanase and laminarase.  

 

The Applicant stated that P. funiculosum strain PF8/403-M is non-toxigenic (i.e. does not produce mycotoxins) and that the conditions used to produce cellulase are not conducive to mycotoxin production, although no evidence was given to substantiate either claim.  Certain publications cited by the Applicant reported the co-occurrence of P. funiculosum in materials that contained mycotoxins, however, other fungi were also present in the samples and therefore it was unclear whether P. funiculosum had actually produced the mycotoxins (see Abdel-Hafez et al 1986; el-Maghraby & Abdel-Sater 1993; Magnoli et al 1998).

 

An independent search of the scientific literature identified some studies suggesting that mycotoxins can be produced by P. funiculosum.  Two isolates of P. funiculosum derived from corn grains produced ochratoxin A and were moderately toxic in a brine shrimp bioassay (50-75% mortality) (Abdel-Mallek et al 1993).  One of three isolates of P. funiculosum isolated from mouldy pecan nuts caused reduced growth rates in chicks (Doupnik & Bell 1971).  A subsequent study found that two isolates derived from weevil-damaged chestnuts were toxic to chicks, causing tremors and mortality (Wells & Payne 1975).  More recent evidence indicated that P. funiculosum does not produce mycotoxins (Kozlovsky et al 1999; Frisvad et al 2004; Labuda & Tančinová 2006).  The European Food Safety Authority (EFSA 2006) reported that P. funiculosum, in general, produces no known mycotoxins except for a single strain that produces secalonic acid.  EFSA concluded that P. funiculosum is not eligible for a Qualified Presumption of Safety (QPS) because it produces ‘many extrolites of unknown structure and biological activity’.

 

Antimicrobial compounds derived from P. funiculosum have been known for some time and include helenine (an antiviral ribonuclear protein) (Shope 1948), funiculosin (an antibiotic) (Ando et al 1969 & 1978) and 4-diazo-3-methoxy-2,5-cyclohexadien-1-one (an antibiotic) (Singh et al 1986).  P. funiculosum strain No. 8974 isolated from soil was reported to produce a novel non-steroidal aromatase inhibitor, TAN-931 (Ishi et al 1991).  In the absence of any information beyond the isolation and basic characterisation of these compounds, it appears that these compounds have found no clinical use in either animal or human medicine.

 

The weight-of-evidence clearly indicates that P. funiculosum strain PF8/403-M is unlikely to be toxigenic.  An added assurance of safety is that the impurity limits provided by the Applicant, which are compliant with international standards, indicated that no P. funiculosum or mycotoxins are present in the final cellulase product.

 


The cellulase protein is also widespread in the environment due to its production by bacteria, fungi and plants.  It is a reasonable expectation that people are already widely exposed to cellulase as a natural part of their diet.  Further, there is a safe history of use of cellulase in the food, animal feed and pharmaceutical industries (TGA 2004).  In particular, cellulase derived from a number of microbial sources is already approved in the Code as a food processing aid.  Cellulase derived from P. funiculosum has GRAS status in the US and Brazil and is sold in Europe for use in brewing.  LAMINEX® C2K contains no impurities of toxicological or biological concern such as mycotoxins or antibiotics.

 

Cellulase derived from P. funiculosum (strain unspecified) had very low acute oral toxicity, with the LD50 in rats greater than 2000 mg/kg bw (McRae 1995).  It was not mutagenic in bacteria (Jones & Gant 1995) and did not cause chromosomal aberrations in human lymphocytes (Adams et al 1995).  There was no evidence of toxicity in rats fed a concentrated liquid derived from P. funiculosum for 90 days; the NOEL was 1000 mg/kg bw/day, the highest dose tested (Jones et al 1998).

 

On the basis that cellulase is a natural part of the diet and that the small concentrations of cellulase (if any) present in the final food would be metabolised as any other dietary protein, it is unnecessary to set an ADI. Therefore the ADI can be considered to be ‘not specified’.

 

Conclusion

 

Based on the available evidence, it is concluded that there are no public health and safety concerns associated with the use of cellulase derived from P. funiculosum strain PF8/403-M as a processing aid.  The ADI can be considered to be ‘not specified’.

 

References

 

Abdel-Hafez SI, Shoreit AA, Abdel-Hafez AI & el-Maghraby OM (1986) Mycoflora and mycotoxin-producing fungi of air-dust particles from Egypt. Mycopathologica 93: 25-32.

 

Abdel-Mallek AY, El-Maraghy SSM & Hasan HAH (1993) Mycotoxin-producing potential of some aspergillus, penicillium and fusarium isolates found on corn grains and sunflower seeds in Egypt. Journal of the Islamic Academy of Sciences 6(3): 189-192.

 

Adams K, Henly SM & Stocker KJ (1995) Cellulase 2000L: Metaphase chromosome analysis of human lymphocytes cultured in vitro. Lab: Huntingdon Research Centre Ltd, Cambridgeshire, England. Report No. RNP 459/951239. Sponsor: Rhone Poulec, ABM Brewing and Enzymes Group, Cheshire, England. Unpublished.

 

Ando K, Suzuki S, Saeki T, Tamura G & Arima K (1969) Funiculosin, a new antibiotic. I. Isolation, biological and chemical properties. The Journal of Antibiotics 22: 189-194.

 

Ando K, Matsura I, Nawata Y, Endo H, Sasaki H, Okytomi T, Saehi T & Tamura G (1978) Funiculosin, a new antibiotic. II. Structure elucidation and antifungal activity. The Journal of Antibiotics 31(6): 533-538.

 

Doupnik B & Bell DK (1971) Toxicity to chicks of Aspergillus and Penicillium species isolated from mouldy pecans. Applied Microbiology 21(6): 1104-1106.

 

EFSA (2006) Annex 6: Assessment of filamentous fungi with respect to a Qualified Presumption of Safety. Available online at: http://www.efsa.eu.int/cs/BlobServer/DocumentSet/Annex6.pdf?ssbinary=true

 

El-Maghraby OM & Abdel-Sater MA (1993) Mycoflora and natural occurrence of mycotoxins in tobacco from cigarettes in Egypt. Zentralbl Mikrobiol 148: 253-264.

 


FAO (2006) General Specifications for Enzymes Used in Food Processing. FAO Food and Nutrition Paper – Monograph 3. Available online at: ftp://ftp.fao.org/docrep/fao/009/a0675e/a0675e00.pdf

 

FAO (2006) Combined compendium of Food Additive Specifications. Monograph 1 - Cellulase from Penicillium funiculosum. Available online at: http://www.fao.org/ag/agn/jecfa-additives/specs/Monograph1/Additive-123.pdf

 

Food Chemicals Codex (2004), National Academy of Sciences, Food and Nutrition Board, Committee on Food Chemical Codex, 5th Edition, National Academy press, Washington DC, pp146-152.

 

Frisvad JC, Smedsgaard J, Larsen TO & Samson RA (2004) Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Studies in Mycology 49: 201-241.

 

Ishi T, Hida T, Ishimaru T, Iinuma S, Sudo K, Muroi M, Kanamaru T & Okazaki H (1991) TAN-931, A novel nonsteroidal aromatase inhibitor produced by Penicillium funiculosum No. 8974. I. Taxonomy, fermentation, isolation, characterisation and biological activities. The Journal of Antibiotics 44(6): 589-599.

 

Jones E & Gant RA (1995) Cellulase 2000L: Bacterial mutation assay. Lab: Huntingdon Research Centre Ltd, Cambridgeshire, England. Report No. RNP 460a/950320. Sponsor: Rhone Poulec, ABM Brewing and Enzymes Group, Cheshire, England. Unpublished.

 

Jones LJ, Bartlett AJ & Brooks PN (1998) Penicillium funiculosum concentrated liquid: Ninety day repeated dose oral (gavage) toxicity study in the rat. Lab: Safepharm Laboratories Ltd, Derby UK. SPL Project No. 854/004. Sponsor: Rhodia Ltd, Stockport, Cheshire, UK. Unpublished.

 

Kozlovsky AG, Marfenina OE, Vinokurova NG, Zhelifonova VP & Adanin VM (1999) Mycotoxin production profiles of the genus Penicillium fungi, isolated from the various environments. Journal of the Japanese Association of Mycotoxicology 48: 37-43.

 

Labuda R & Tančinová D (2006) Fungi recovered from Slovakian poultry feed mixtures and their toxinogenicity. Annals of Agricultural and Environmental Medicine 13: 193-200.

 

Losada E, Hinojosa M, Moneo I, Dominguez J, Diez Gomez ML & Ibanez MD (1986) Occupational asthma caused by cellulase. Journal of Allergy & Clinical Immunology 77(4): 635-9

 

Magnoli C, Dalcero AM, Chiacchiera SM, Miazzo R & Saenz MA (1998) Enumeration and identification of Aspergillus group and penicillium species in poultry feed from Argentina. Mycopathologica 142: 27-32.

 

McRae L (1995) Cellulase 2000L: Acute oral toxicity to the rat. Lab: Huntingdon Research Centre Ltd, Cambridgeshire, England. Report No. RNP 461a/950521/AC. Sponsor: Rhone Poulec, ABM Brewing and Enzymes Group, Cheshire, England. Unpublished.

 

Shope RE (1948) Therapeutic activity of a substance from Penicillium funiculosum Thom against swine influenza virus infection of mice. American Journal of Botany 35: 803.

 

Singh PD, Johnson JH, Aklonis CA & O’Sullivan J (1986) SQ 30,957, A new antibiotic produced by Penicillium Funiculosum. Taxonomy, fermentation, isolation, structure determination, synthesis and antibacterial activity. The Journal of Antibiotics 34(8): 1054-1058.

 

Tarvainen K, Kanerva L, Tupasela O, Grenquist-Norden B, Jolanki R, Estlander T & Keskinen H (1991) Allergy from cellulase and xylanase enzymes. Clinical & Experimental Allergy 21: 609-615.

 

TGA (2004) Complementary Medicines Evaluation Committee. 47th Meeting. Department of Health & Ageing. Available online at: http://www.tga.gov.au/DOCS/pdf/cmec/cmecmi47.pdf

 

Wells JM & Payne JA (1975) Toxigenic Aspergillus and Penicillium isolates from weevil-damaged chestnuts. Applied Microbiology 30(4): 536-540.

 


WHO (1987) Evaluation of Certain Food Additives and Contaminants. 31st Report of the Joint FAO/WHO Expert Committee on Food Additives. Technical Report Series 759.

 

WHO (1988) Specifications for the identity and purity of certain food additives. FAO Food and Nutrition Paper No 38.

 

 


Attachment 4

 

Food Technology Report

 

A1011 – Cellulase from Penicillium funiculosum as a processing aid (Enzyme)

 

Summary

 

Danisco A/S submitted an Application to amend Standard 1.3.3 – Processing Aids of the Australia New Zealand Food Standards Code (the Code) to permit a new cellulase enzyme preparation (EC number 3.2.1.4).  This enzyme is sourced via a fermentation process from a non-toxigenic and non-pathogenic classical strain of Penicillium funiculosum. 

 

This enzyme preparation and its source are requested to be permitted as an approved processing aid in the Table to clause 17 – Permitted enzymes of microbial origin of Standard 1.3.3.  The approval of the cellulase preparation from this source would provide an alternative to the currently permitted sources of cellulase already in the Code, including Aspergillus niger, Trichoderma reesei and Trichoderma viride

 

This cellulase enzyme preparation is proposed to be used in the brewing and distilling industries.  In the brewing industry, the enzyme is claimed to reduce the viscosity of the wort and improve the separation of the wort from the spent grain. 

 

In the distilling industry this enzyme is claimed to reduce the mash viscosity and ultimately to improve the overall processing efficiency.  The use of this cellulase enzyme preparation would be advantageous and is technologically justified in the brewing and distilling industries.

 

Introduction

 

The number of food enzymes and the microbial species from which they are produced has greatly expanded in recent times, partially in response to the constantly evolving and expanding requirements of the international food industry (Pariza and Johnson, 2001).  Microbial enzyme preparations have been widely used for a variety of purposes in the production of numerous food products for many years.  Their practical application in fermented products dates back many centuries, long before the nature and function of enzymes or even the microorganisms themselves, were known or understood (Bhat, 2000).  Cellulases and related enzymes have great biotechnology potential and are used in the food, brewery, distilling and wine industries as well as in animal feed, agriculture, paper and pulp industries, textile and laundry and in other related applications (Beguin and Aubert, 1994; Bhat, 2000; Bayer et al., 2007). 

 

Danisco A/S submitted an Application to amend Standard 1.3.3 – Processing Aids to permit a new cellulase enzyme preparation (EC number 3.2.1.4), produced by fermentation from a non-genetically modified, non-toxigenic and non-pathogenic strain of P. funiculosum as a processing aid.  Various cellulase enzyme preparations (EC 3.2.1.4) are currently listed as permitted processing aids in the Table to clause 17 of the code, these enzyme preparations are obtained from the following sources; A. niger, T. reesei and T. viride

 

The Applicant proposes that this cellulase enzyme preparation can be used as a processing aid in the brewing and distilling industries. In the brewing industry the enzyme is claimed to reduce the viscosity of the wort and to improve the separation of the wort from the spent grain.  In the distilling industry, use of the enzyme is claimed to reduce the viscosity of the mash and improve the overall processing efficiency. 


This commercial cellulase preparation is claimed to comply with internationally recognised specifications for the production of enzymes, specifically the specifications of the Joint FAO/WHO Expert Committee on Food Additives (JECFA, 2006) and Food Chemicals Codex (Committee on Food Chemical Codex, 2004) specifications for enzyme preparations.  Detailed specifications for cellulase from P. funiculosum have also been revised and published at the 55th JECFA meeting (JECFA, 2000).

 

Characterisation of the enzyme

 

This cellulase enzyme preparation sourced from P. funiculosum is claimed to be a mixture of a number of cellulose degrading enzymes.  Its action is a result of the synergistic effect the main and side activities of all the individual enzymes present including endoglucanases, cellobiohydrolases and beta glucosidase.  The enzyme is standardised on the basis of its cellulase activity on carboxymethylcellulose (CMC).

 

Reaction

 

The main reaction of cellulase is the endohydrolysis of 1, 4-beta-D-glycosidic linkages in polysaccharides such as cellulose, lichenin and cereal beta-glucans, yielding beta-dextrins (JECFA 2000; IUBMB, 2001). The Applicant claims it will also hydrolyse 1,4-linkages in

β-D-glucans that also contain 1,3-linkages.

 

Other minor activities involving the other minor enzymes present include beta glucosidases enzymes working via the endohydrolysis of 1,3- or 1,4-linkages in ß-D-glucans; xylanase catalysing the endohydrolysis of 1,4-ß-D-xylosidic linkages in xylans; and glucan endo-1,3-ß-D-glucosidase catalysing the hydrolysis of 1,3-ß-D-glucoosidic linkages in 1,3-ß-D-glucans.

 

Identity, purity and specifications of the enzyme

 

(a)     Identity

 

The common name of the enzyme derived from P. funiculosum is ‘cellulase’.

 

Other names used include endoglucanase, endo-1,4-betaglucanase, carboxymethylcellulase, endo-1,4-beta-D-glucanase, beta-1,4-glucanase, beta-1,4-endoglucanhydrolase, celludextrinase and avicelase (IUBMB, 2001).  This enzyme is produced via fermentation utilising a selected classical strain of P. funiculosum. 

 

·                Commercial name:       LAMINEX® C2K (LAMINEX ® C2000)

·                Systematic name:         1,4-(1,3;1,4)-ß-D-glucan 4-glucanohydrolase

·                EC number:                  3.2.1.4

·                CAS number:                9012-54-8

 

This cellulase enzyme preparation derived from P. funiculosum also contains other minor enzyme activities that are essential to the applications of this enzyme preparation.  These include ß -glucanase (EC 3.2.1.6), xylanase (EC 3.2.1.8), and glucan endo-1,3-ß-D-glucosidase (EC3.2.1.39). 

 

(b)     Purity

 

Appropriate Good Manufacturing Practices (GMP) controls are used in the manufacture of cellulase preparation from P. funiculosum with the following specification for impurities and microbial limits listed in Table 1; these fall well within the JECFA specification (JECFA 2000)


This is to ensure the final treated food product does not contain any impurities of a hazardous or toxic nature. 

 

JECFA has evaluated the cellulase preparation from P. funiculosum at the 31st JECFA meeting (1987).  The specifications have been published in Food and Nutrition Paper 38 (1988) and have been revised at the 55th JECFA meeting (2000).  The revised specifications have been published in JECFA specification FNP 52 Addenda 8 (2000). 

 

This commercial cellulase preparation complies with internationally recognised specifications for the production of enzymes, specifically with the Food Chemicals Codex  (5th Edition, 2004) and the FAO Food and Nutrition Paper, Monograph 3 - ‘General Specification for Enzyme Preparations used in Food Processing’,  FAO Food and Nutrition Paper (JECFA, 2006).  These specifications referenced are both primary sources of specifications listed in clause 2 of Standard 1.3.4 – Identity and Purity. 

 

Table 1:  Specification for Cellulase preparation from P. funiculosum

 

Criteria

 

Specification for cellulase preparation from

P. funiculosum

 

Metals

 

 

Heavy metals as Pb

less than 30 mg/kg

 

Arsenic

less than 3 mg/kg

 

Cadmium

less than 0.50 mg/kg

 

Mercury

less than 0.50 mg/kg

 

Lead

Not more than 5 ppm

 

Microbiological

 

Total viable count

less than 5 x104 /mL

Total coliforms

less than 30 /mL

E. coli

absent in 25 mL

Salmonella

absent in 25 mL

Antibiotic activity

negative by test

Production strain

Absent

Other Assays

 

Mycotoxins

Negative

Minimum activity

3150 CMC U/g*

Physical Properties

 

pH

3.7–4.2

Appearance

non viscous, clear, light brown liquid

Temperature optimum

approximately 55ºC

Thermal stability

not stable above 60ºC

pH optimum

approximately pH 5

pH stability

4–7

*The activity of the cellulase enzyme preparation is expressed in carboxymethylcellulose activity units (CMC U). One CMC unit of activity liberates 1 μmol of reducing sugars (expressed as glucose equivalents) in one minute under specific assay conditions (50°C and pH 4.8).

 

Enzyme production

 

The cellulase enzyme preparation is produced via a submerged fermentation process utilising appropriate substrates and nutrients under good manufacturing practices. 

 

After the fermentation process is complete, the biomass is removed by centrifugation and filtration processes.  The remaining fermentation broth, containing the enzyme, is subsequently concentrated.  This concentrated enzyme solution is standardised and stabilised using appropriate additives such as sodium benzoate.  Filtration is used as a final purification step for the cellulase preparation. 


Throughout the manufacturing process, cellulase is monitored and carefully controlled by analytical and quality assurance procedures which ensure that the final product complies with the appropriate specifications as listed above and is suitable for use as a processing aid in food processing applications. 

 

Source organism

 

The source microorganism is claimed to be a food grade non pathogenic and non toxigenic strain of P. funiculosum developed via classical strain improvements only and is not genetically modified.

 

Technological function of the enzyme / Applications

 

Active research on the uses of cellulase enzymes was being conducted as early as the 1950’s on their potential to convert celluloses into glucose and soluble sugars and these enzyme preparations have been actively researched since this time (Schwardt, 1990; Bhat, 2000).  Cellulases have been shown to have enormous potential for use in food biotechnology arenas.  Uses for these cellulose degrading enzymes include the extraction and clarification of fruit and vegetable juices, the production of fruit nectars and purees, in the improvement of the sensory properties of fruits and vegetables extracts, extraction of olive oil and to improve the quality of bakery products.  Cellulases have also been extensively utilised in the winery, brewing and distilling industries (Bhat, 2000). 

 

The biological degradation of cellulose has been thoroughly reviewed by a number of authors and the literature generally indicates that the enzymatic hydrolysis of cellulose by cellulase enzymes are highly specific reactions (Gams, 1976; Schwardt, 1990; Beguin and Aubert, 1994; Bhat, 2000).  Cellulase enzyme preparations are primarily utilised to catalyse the enzymatic hydrolysis of cellulose.  Cellulase enzyme preparations are often a complex mixture of several enzymes all intimately involved in the hydrolysis of cellulose (Mussatto et al., 2006)The performance of this particular enzyme preparation is a result of the synergistic effects of cellulase, endoglucanases, cellobiohydrolases and beta glucosidase enzymes present.  

 

A study by Karboune et al., (2008) investigated the characterisation of selected celluloytic activities of a multi-enzymatic preparation produced from P. funiculosum, and found an interesting feature was the synergistic action of the enzymes working together for the hydrolysis of native cellulose into individual glucose units.  In this study P. funiculosum was noted as being widely recognised as an important source of highly active multi-cellulolytic preparation systems capable of degrading cellulose in a number of practical situations.

 

(a)     Beer

 

Beer production has an ancient history and has been conducted for many centuries. Essentially the production of beer involves brewing and fermenting the starches derived from cereals grains.  Grain undergoes malting followed by the addition of water and the preparation and fermentation of the wort.  Barley is the most common cereal used for the production of beer although wheat, corn, and rice are also widely used.  Beer is often flavoured with hops, which is utilised to impart a bitter taste to the final product.  Other adjuncts such as sugars may also be added.  The main processes involved in beer production include milling to reduce the size of the dry malt in order to increase availability of carbohydrates, mashing where water is added to the malt, lautering or filtration where spent grains are removed form the wort, boiling the wort with flavouring hops, fermentation of the wort liquor, maturation, conditioning, filtration and packaging of the final product.

 


Enzyme technology and interaction can play a key role in the production of beer.  The addition of cellulases and other polysaccaridases are known to both improve the quality of the final product as well as increase overall productivity of the manufacturing process. 

 

Endogenous enzyme production generally occurs naturally during the malting stage of beer production; which involves the barley grain (and/or other cereal grains) being held under conditions which encourage the grain to germinate.  The biosynthesis and activation of endogenous enzymes (e.g. alpha and beta amylases, carboxypeptidase and beta glucanase) serve to hydrolyse the seed’s carbohydrate reserves.  In an ideal world this endogenous mixture of enzymes should work synergistically under optimal conditions to produce consistent high quality malted wort liquid.  In reality however, due to seasonal variations in the grain, poor harvests and/or using a combination of different cultivars together, the wort may potentially contain un-malted grains along with an excess amount of high molecular weight polysaccharides such as beta glucans (Bhat, 2000).  This excess of beta glucans has the potential to increase mash viscosity and cause haze formation, poor dewatering, low yields, slow run off time, and lead to poor filterability of the wort and the development of an unwanted haze in the final beer.  The addition of a commercial cellulase enzyme preparation (specifically containing beta glucanases) at the mashing stage can serve to hydrolyse excess beta glucans and ultimately reduce the viscosity of the wort (Bhat, 2000). 

 

The Applicant states that this cellulase enzyme preparation will be used during the mashing stage to reduce the viscosity of the wort and subsequently improve the separation of the wort from the spent grain.  The cellulase enzyme preparation degrades beta glucans and arabinoxylans which are extracted during the mashing process and thereby reduces the viscosity of the wort and improves the separation of the wort from the spent grains.  The level of enzyme use proposed is 10-100 mg of active enzyme per kg of cereal.

 

(b)     Distilling

 

The distilling process involves four main steps including milling to reduce the size of the malted grain, mashing where water is added to the malt, fermentation and the final distillation of the spirit.  The most common grains used in distilling beverages include wheat and barley.

 

The Applicant states that this cellulase enzyme preparation will be added during the mashing step to degrade non-starch polysaccharides such as beta glucans and arabinoxylans, resulting in a decrease in mash viscosity, more efficient processing, including a reduction in the amount of fuel required due to an improvement in heat transfer, and a potential increase in alcohol yields.  The level of enzyme use proposed by the Applicant is between 10–100 mg of active enzyme per kg of grist. 

 

Allergenicity

 

Given the manufacturing process and ingredients described above and within the Application, no allergenic materials (as listed in the Code on Table to clause 4 of Standard 1.2.3) are present in the manufacture or final enzyme preparation.

 

The cellulase enzyme itself is not considered to be allergenic.

 


Conclusion

 

The use of the cellulase enzyme preparation, sourced from P. funiculosum, as a processing aid is technologically justified to catalyse the enzymatic hydrolysis of cellulose and other polysaccharides.  The use of the cellulase enzyme preparation would be advantageous and technologically justified in the brewing and distilling industries.

 

References

 

Bayer, E.A., Lamed, R. and Himmel, M.E. (2007) The potential of cellulases and cellulosomes for cellulosic waste management. Current Opinion in Biotechnology 18(3):237-245.

Beguin, P. and Aubert, J.P. (1994) The Biological Degradation of Cellulose. Fems Microbiology Reviews 13(1):25-58.

Bhat, M.K. (2000) Cellulases and related enzymes in biotechnology. Biotechnology Advances 18(5):355-383.

Cao, Y. and Tan, H.M. (2002) Effects of cellulase on the modification of cellulose. Carbohydrate Research 337(14):1291-1296.

Committee on Food Chemical Codex. (2004) Food Chemical Codex. National Academy of Sciences, Food and Nutrition Board, National Academy Press, Washington DC. 5th Edition.

Gams, T.C. (1976) [Applications of enzymes in food technology.]. Gordian: 76 (7/8) 210, 212, 214, 216, 218, 219; (9) 254, 256 76(7/8):210.

IUBMB (2001) EC 3.2.1.4. http://www.chem.qmul.ac.uk/iubmb/enzyme/EC3/2/1/4.html. Accessed on 22 September 2008.

JECFA (2000) Cellulase from Penicillium funiculosum.  Joint FAO/WHO Expert Committee on Food Additives, 55th session, FNP 52 Add 8.  http://www.fao.org/ag/agn/jecfa-additives/specs/Monograph1/Additive-123.pdf. Accessed on 16 September 2008.

JECFA (2006) Compendium of Food Additive Specifications - General specifications and considerations for enzyme preparations used in food processing. Joint FAO/WHO Expert Committee on Food Additives. FAO Food and Nutrition Paper Monograph 3, 67th session, 63-67. ftp://ftp.fao.org/docrep/fao/009/a0675e/a0675e00.pdf. Accessed on 16 September 2008.

Karboune, S., Geraert, P.A. and Kermasha, S. (2008) Characterization of selected cellulolytic activities of multi-enzymatic complex system from Penicillium funiculosum. J Agric Food Chem 56(3):903-909.

Mussatto, S.I., Dragone, G. and Roberto, I.C. (2006) Brewers' spent grain: generation, characteristics and potential applications. Journal of Cereal Science 43(1):1-14.

Mussatto, S.I., Fernandes, M., Milagres, A.M.F. and Roberto, I.C. (2008) Effect of hemicellulose and lignin on enzymatic hydrolysis of cellulose from brewer's spent grain. Enzyme and Microbial Technology 43(2):124-129.

Pariza, M.W. and Johnson, E.A. (2001) Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul.Toxicol Pharmacol. 33(2):173-186.

Schwardt, E. (1990) Production and use of enzymes degrading starch and some other polysaccharides. Food Biotechnology: 4 (1) 337-351 4(1):337-351.

 



[1] Bhat, M.K. (2000) Cellulases and related enzymes in biotechnology. Biotechnology Advances 18(5):355-383.

 

[2] JECFA (2006) Compendium of Food Additive Specifications - General specifications and considerations for enzyme preparations used in food processing. Joint FAO/WHO Expert Committee on Food Additives. FAO Food and Nutrition Paper Monograph 3, 67th session, 63-67. ftp://ftp.fao.org/docrep/fao/009/a0675e/a0675e00.pdf. Accessed on 16 September 2008.

 

[3] Committee on Food Chemical Codex (2004) Food Chemical Codex. 5th Edition. National Academy of Sciences, Food and Nutrition Board, National Academy Press, Washington DC.

 

[4] In May 2008, the Australia and New Zealand Ministerial Council finalised the Policy Guideline on Addition to Food of Substances other than Vitamins and Minerals.  This includes policy principles in regard to substances added for technological purposes such as food additives and processing aids.  FSANZ has paid regard to each of these principles in assessing this Application.