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WHO FOOD ADDITIVES SERIES: 52

MIXED XYLANSE, beta-GLUCANASE ENZYME PREPARATION
PRODUCED BY A STRAIN OF HUMICOLA INSOLENS

First draft prepared by

Mrs I.M.E.J. Pronk
Centre for Substances and Integrated Risk Assessment, National Institute for Public Health and the Environment, Bilthoven, Netherlands

and

Dr C. Leclercq
National Research Institute for Food and Nutrition, Rome, Italy

Explanation

Biological data

Biochemical aspects

Toxicological studies

Acute toxicity

Short-term studies of toxicity

Long-term studies of toxicity and carcinogenicity

Genotoxicity

Reproductive toxicity

Observations in humans

Dietary intake

Comments

Evaluation

References

1. EXPLANATION

The mixed beta-glucanase and xylanase preparation under evaluation is produced by fed-batch, submerged, pure culture fermentation of a strain of Humicola insolens that is non-pathogenic and non-toxigenic. This enzyme mixture has not been evaluated previously by the Committee.

The enzyme preparation contains two main activities, beta-glucanase and xylanase, and several secondary activities, including cellulase, hemicellulase, pentosanase and arabinase. The preparation is used in beer brewing to hydrolyse beta-glucans, pentosans and other gums. This reduces the viscosity of the solution and thereby increases the filtration rate of both wort and beer and improves beer clarity. The production strain has been selected for improved enzyme production. The manufacturing procedure comprises a fermentation process, a purification process, a formulation process and finally quality control of the finished product. The cell mass and other solids are separated from the broth by filtration or centrifugation. Ultrafiltration and/or evaporation are used for concentration and further purification. The liquid enzyme concentrate (LEC) is then stabilized, formulated and standardized by the addition of sorbitol, glycerol, and potassium sorbate.

beta-Glucanase hydrolyses the barley beta-glucans under formation of mono- or oligomers. Xylanase hydrolyses xylosidic linkages in the arabinoxylan backbone resulting in a depolymerization of the arabinoxylans into smaller oligosaccharides.

The evaluated enzyme preparation, known as "Ultraflo L", is used in the food industry as a processing aid. It is mainly used in the brewing of beer, but may also be used in the alcohol industry, to increase the filtration rate of both wort and beer, and prevent the formation of haze. The recommended dosage is 200 g of the enzyme preparation per 1000 kg of total raw materials, but is dependent on the composition of the raw material. The mixed beta-glucanase/xylanase preparation is also marketed within the European Union as a feed-additive under the name of "Pentopan/Biofeed Plus."

The mixed enzyme preparation is standardized on the main activity, beta-glucanase. It has a typical activity of 45 fungal beta-glucanase (FBG) per g. In addition, the enzyme preparation contains approximately 470 farbe xylanase units (FXU) per g. The typical composition of Ultraflo L is as follows: total organic solids (TOS), approximately 5%; water, approximately 40%; glycerol, approximately 28%; sorbitol, approximately 27%; potassium sorbate, approximately 0.2%.

Toxicological studies have been performed with an LEC (batch PPC 5569), omitting formulation, stabilization and standardization. In these studies, batch PPC 5569 was named Pentopan/Biofeed Plus; the composition of batch PPC 5569 is given in Table 1.

Table 1. Composition of mixed enzyme preparation

 

Batch PPC 5569

Enzyme activity,
FBG/g

45

FXU/g

644

EGU/g

82.1

TOS (% w/w)

6.1

Density (g/ml)

1.021

From Jensen (2002); Pedersen (2002)

EGU, units of endo-cellulase activity; FXU, farbe xylanase
units; FBG, fungal beta-glucanase; TOS, total organic solids

2. BIOLOGICAL DATA

2.1 Biochemical aspects

No information was available.

2.2 Toxicological studies

2.2.1 Acute toxicity

No information was available.

2.2.2 Short-term studies of toxicity

Rats

Groups of 10 male and 10 female Wistar rats (body weight: males, 155.9 156.3 g; females 123.1123.8 g) received water containing the mixed enzyme preparation (batch PPC 5569, also known as Pentopan/Biofeed Plus) at a dose of 0, 1, 3.3, or 10.2 g/kg bw per day (equivalent to 0, 644, 2125, or 6569 FXU/kg bw per day, 0, 45, 149, or 459 FBG/kg bw per day, and 0, 0.06, 0.20, or 0.62 g/kg bw per day of TOS) by oral gavage for 13 weeks. The study was performed according to OECD test guideline 408 (1981), and was certified for compliance with good laboratory practice (GLP) and quality assurance. All animals were checked daily for mortality and moribundity, general condition and behaviour. Ophthalmoscopy was performed before treatment for all rats in all groups and at the end of treatment for all rats in the control group and in the group receiving the highest dose. Body weight and food consumption were recorded weekly and food conversion efficiency was calculated. Food and water were freely available. During week 13, haematology, clinical chemistry and urine analysis were performed for all animals. Absolute and relative (to body weight) weights of eight organs were determined. All animals were examined macroscopically. Microscopy was performed on about 40 organs and tissues from all animals in the control group and in the group receiving the highest dose. In addition, kidneys, liver and lungs from all animals in the groups receiving the low and intermediate doses, and all macroscopically abnormal tissues were examined microscopically.

No effects on survival or behaviour were seen. Ophthalmoscopy was normal. Body-weight gain was normal. Food consumption of males at the highest dose was slightly, but significantly, decreased during the first few weeks of the study. Food conversion did not show significant changes. There were no abnormalities in haematological or clinical chemistry parameters or in urine analysis. The renal clearance test showed a significant decrease in the density of the urine of males at the low and intermediate doses. Urinary volume was slightly, but not significantly, higher in males in all treated groups, but no dose-reponse relationship was seen. Organ weights were normal and macroscopy and microscopy did not reveal any effects related to treatment. It can be concluded that in this 13-week study in rats treated orally, the NOEL for batch PPC 5569 was the highest dose, 10.2 g/kg bw per day (equivalent to 6569 FXU plus 459 FBG/kg bw per day and 0.62 g of TOS/kg bw per day) (Lina, 1997; Pedersen, 2002).

2.2.3 Long-term studies of toxicity and carcinogenicity

No information was available.

2.2.4 Genotoxicity

The results of two assays for genotoxicity in vitro with batch PPC 5569 (Pentopan/Biofeed Plus) are summarized in Table 2. Both studies followed OECD test guidelines, 471 (1983) and 473 (1983), respectively, and were certified for compliance with GLP and quality assurance.

Table 2. Results of studies of genotoxicity with batch PPC 5569 (Pentopan/Biofeed Plus)

Endpoint

Test object

Concentration

Results

References

In vitro

 

 

 

 

Reverse mutation

S. typhimurium TA98, TA100, TA1535, TA1537

31310 000 g/ml Solvent: sterile water

Negativea

Pedersen (1996); Pedersen (2002)

Chromosomal aberration

Human lymphocytes

2450, 3500, and 5000 g/ml in sterile water

Negativeb

Marshall (1996); Pedersen (2002)

a

In the presence and absence of metabolic activation from S9, no cytotoxicity was seen. Owing to the presence of free amino acids, e.g. histidine, in the mixed beta-glucanase/xylanase preparation, the growth of Salmonella strains requiring histidine was significantly increased after direct-plate incorporation. Therefore, the Salmonella strains were exposed to Pentopan/Biofeed Plus in in a phosphate-buffered nutrient broth in liquid culture ("treat-and-plate assay") for 3 h, at six concentrations, the highest dose being 10 mg/ml. After incubation the test substance was removed by centrifugation prior to plating

b

In the presence and absence of metabolic activation from S9. In the absence of S9, cells were exposed continuously for 20 h and then harvested (mitotic inhibition, 59% at 5000 mg/ml); in the presence of S9, cells were exposed for 3 h and harvested 17 h later (mitotic inhibition, 24% at 5000 mg/ml). In the second experiment, in the absence of S9, cells were exposed for 20 h, and for 44 h at the highest dose, and then harvested (mitotic inhibition, 37% and 32% after 20 h and 44 h, respectively, at 5000 mg/ml). In the presence of S9, cells were exposed for 3 h and harvested 17 or 41 h later (mitiotic inhibition, 4% and 0% respectively, at 5000 g/ml) S9, 9000 g supernatant of rat liver homogenate

2.2.5 Reproductive toxicity

No information was available.

2.3 Observations in humans

No information was available.

3. DIETARY INTAKE

A complete and comprehensive list of enzymes and their uses in food manufacturing in the European Union is not available, but an inventory of enzyme use in nine Member States was compiled for scientific cooperation (SCOOP) Task 7.4 (European Commission, 2000). It was reported that the specific enzyme preparation under evaluation was in use in beer production in Denmark.

Although it is not necessary to perform intake estimates since enzymes or other organic solids from the enzyme preparation are not expected to persist in the final product, very conservative estimates of daily intakes were made. Recommended dosage and TOS content of the enzyme preparation were provided by the sponsor (Jensen, 2002).

A "worst-case" scenario for intake from beer was estimated on the basis of the following assumptions:

According to the budget method, the upper physiological intake of liquid is 100 ml/kg bw per day, or 6 l for a 60-kg person (Hansen, 1979). A "worst-case" scenario is that of ingestion at 50 ml/kg bw per day of beer, corresponding to an intake of 0.085 mg of TOS/kg bw per day (200 5.1% / 6 0.05), i.e. 5.1 mg of TOS per day for a 60-kg person.

The "worst case" scenario for intake from alcohol in spirits was estimated on the basis of the following assumptions:

A "worst-case" scenario is that of ingestion of 0.5 l of spirit, leading to an intake of 0.04 mg of TOS/kg bw per day ([0.5/60] 0.35 5% 200 1000/640), i.e.

2.7 mg of TOS per day for a 60-kg person.

Assuming that all TOS would persist in the final products, very high intakes of beer and spirits lead to an estimated cumulative daily intake of 0.125 mg of TOS/kg bw. Compared with the NOEL of 0.62 g of TOS/kg bw per day in the 13-week study of oral toxicity, the margin of safety is nearly 5000.

4. COMMENTS

Toxicological studies were conducted on the LEC. The materials added to the LEC upon stabilization, formulation and standardization have either been evaluated previously by the Committee or are common food constituents and do not raise safety concerns.

In a 13-week study in rats, no significant treatment-related effects were seen when the LEC was administered orally by gavage at doses of up to and including 10.2 g/kg bw per day. Therefore this highest dose (equivalent to an intake of 0.62 g of TOS/kg bw per day) was the NOEL. The LEC was not active in an assay for mutagenicity in bacteria in vitro nor in an assay for chromosomal aberrations in mammalian cells in vitro.

The enzyme preparation is added during the mashing process of beer-making and the enzymes are denatured and inactivated during the subsequent wort-boiling stage. The beer filtration process is likely to remove the denatured enzymes along with other insoluble materials. The preparation may also be used in the spirits industry; again, in this case, no enzymes or other organic solids are expected to be carried over into the final product because ethanol is removed by distillation from the fermentation mash containing the enzyme preparation. In conclusion, no residual enzymes are expected to be present in food processed using this enzyme preparation. The Committee is not aware of any other uses for this enzyme mixture in which the enzymes might persist in the final product.

Very conservative estimates of daily intakes were performed on the basis of the assumption that all TOS would persist in final products. These gave an estimated daily intake of 5.1 mg of TOS/day (equivalent to 0.085 mg/kg bw per day) for beer and 2.7 mg of TOS/day (equivalent to 0.04 mg/kg bw per day for a 60-kg person) for spirits. Compared with the NOEL of 0.62 g of TOS/kg bw per day in the 13-week study of oral toxicity, the margin of safety is nearly 5000.

5. EVALUATION

The Committee allocated an ADI "not specified" to mixed beta-glucanase/xylanase from the production strain H. insolens, used in the applications specified and in accordance with good manufacturing practice.

6. REFERENCES

European Commission (2000) Report on task for scientific cooperation (SCOOP). Report of experts participating in Task 7.4. Study of the enzymes used in foodstuffs and collation of data on their safety (http://www.europa.eu.int/comm/food/fs/scoop/index_en. html).

Hansen S.C. (1979) Conditions for use of food additives based on a budget for an acceptable daily intake. J. Food Protect., 42, 42934.

Jensen, B.R. (2002) Mixed beta-glucanase, xylanase enzyme preparation, produced by a strain of Humicola insolens. UltrafloTM L. Unpublished report No. 2002-3774901 from Novozymes A/S, Bagsvaerd, Denmark. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

Lina, B.A.R. (1997) Sub-chronic (13-wk) oral toxicity study with Pentopan/Biofeed Plus in rats. Unpublished report No. V96.898 from TNO Nutrition and Food Research, Zeist, Netherlands. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

Marshall, R. (1996) Pentopan/Biofeed Plus: Induction of chromosome aberrations in cultured human peripheral blood lymphocytes. Unpublished report No. 665/139 from Corning Hazleton (Europe), Harrogate, England. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

Pedersen, P.B. (1996) Pentopan/Biofeed Plus, (batch number: PPC 5569): Testing for mutagenic activity with Salmonella typhimurium TA98, TA1537, TA100 and TA1535 in a treat and plate assay. Unpublished report No. 968084 from Novo Nordisk A/S, Bagsvaerd, Denmark. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.

Pedersen, P.B. (2002) Summary of toxicity data. Ultraflo. Unpublished report No. 2002-0162101 from Novozymes A/S, Bagsvaerd, Denmark. Submitted to WHO by Novozymes A/S, Bagsvaerd, Denmark.



    See Also:
       Toxicological Abbreviations