Danisco Specialities

TM
Natamax and Nisaplin®

- natural antimicrobials from

Danisco

Effective preservatives for fruit juice

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 Fruit juice spoilage
Danisco Specialities

Mould spoilage due to heat-resistant moulds such

as Byssochlamys and other species may occur in
pasteurised fruit juice

Yeast cause spoilage in unpasteurised juice,

but also in pasteurised juice due to post processing
contamination. Certain yeasts may survive milder
heat treatments

Many yeasts can grow at chill temperatures

The xerophilic yeast Zygosaccharomyces rouxii has

been implicated in spoilage of filling systems for
juice products
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 Heat – resistant moulds
Danisco Specialities

Moulds of the genus Byssochlamys

are associated with spoilage of heat-
processed acid foods

Byssochlamys is found worldwide -

associated with spoilage of fruit
juices, canned/bottled soft fruit and
fruit gel baby foods

The fungus can survive the normal

pasteurisation temperatures that are
used for most fruit and fruit products

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3
 Byssochlamys – the problem
Danisco Specialities

Found in soil - contaminates fruit as well as

containers used to transport/process the fruit

Some strains can produce patulin, a potent

mycotoxin

Unusually tolerant of reduced oxygen conditions

and/or elevated levels of carbon dioxide

Can appear to grow anaerobically - producing

CO2, causing swelling and spoilage of the product

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 Byssochlamys – the solution
Danisco Specialities

Addition of NatamaxTM at a level of 25 – 50

mg/litre

prior to pasteurisation

inhibits the growth of Byssochlamys

and other heat-resistant yeasts or moulds

that may survive heat processing

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5
 Extension of shelf life in orange juice
inoculated with Byssochlamys fulva spores
Danisco Specialities

Incubation at 25 oC. Effect of 2 different heat treatments.

45

40 70 oC for 10 minute
35
Days until visible spoilage

95 oC for 1 minute
30

25

20

15

10

0
0 ppm 2.5 ppm 5 ppm 10 ppm 20 ppm

01.02
Natamycin

6
 Yeasts – the problem
Danisco Specialities

Yeasts are major causes of spoilage of fruit

juices because of their high acid tolerance
and ability to grow anaerobically. Some may
produce ascospores capable of surviving
pasteurisation

Common juice spoilage species include:

Candida parapsilosis
Debaryomyces hansenii
Hanseniaspora uvarum
Rhodotorula mucilaginosa
Saccharomyces cerevisiae
S. exiguus (Torulopsis holmii)
Zygosaccharomyces bailii
01.02
Z. rouxii

7
 Yeasts – the solution
Danisco Specialities

Research in our laboratories shows that addition of NatamaxTM can

significantly increase the shelf-life of fruit juices contaminated with
spoilage yeasts.

For example, in yeast challenge studies

addition of 20 ppm NatamaxTM
increased shelf life at ambient temperature
by 30 to > 50 days.

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 Efficacy of NatamaxTM in controlling yeast
spoilage of unpasteurised apple juice.
Graph shows extension of shelf-life by various natamycin concentrations.
Danisco Specialities

60
D a y s u n til v is ib le s p o ila g e

50

40

30

20

10

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24

25
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Natamycin (ppm) 0 1.25 2.5 5 10

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 Comparison of natamycin (as Natamax™) and sorbate
for control of yeast spoilage in orange juice (pH 3.5)
Danisco Specialities

Days until visible growth

Inoculum in unpasteurised orange juice at 25 oC

Yeast CFU/ ml Control 1.25 ppm 2.5 ppm 300 ppm

natamycin natamycin sorbate

Hanseniaspora uvarum 3.2 x 103 5 6 > 50 > 50

CBS5074
Saccharomyces cerevisiae 1.3 x 103 5 > 50 > 50 6
ATCC9763
S. exiguus CBS1514 2.4 x 103 5 > 50 > 50 7

Zygosaccharomyces bailii 7.0 x 102 5 > 50 > 50 7

CRA229
Z. rouxii CBS1640 1.7 x 103 5 9 > 50 > 50

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 Effect of heat treatment on natamycin and sorbate efficacy
Danisco Specialities

Strain Heat treatment Days until growth observed in orange juice at 25 oC

Natamycin levels (mg/litre) Sorbate
0 1.25 2.5 5 300 mg/litre

H. uvarum None 5 6 > 50 > 50 > 50

70 oC for 10 min 2 8 > 50 > 50 > 50
95 oC for 1 min 2 9 > 50 > 50 > 50
S. None 5 6 > 50 > 50 6
cerevisiae 70 oC for 10 min 2 - > 50 > 50 14
95 oC for 1 min 2 13 > 50 > 50 13
S. exiguus None 5 > 50 > 50 > 50 7
70 oC for 10 min 5 8 33 > 50 19
95 oC for 1 min 2 6 7 > 50 14
Z. bailii None 5 > 50 > 50 > 50 7
70 oC for 10 min 5 > 50 > 50 > 50 9
95 oC for 1 min 6 13 28 28 9
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 Effect of heat treatment on natamycin efficacy
Danisco Specialities

Natamycin activity is slightly reduced as a result of pasteurisation

treatments due to heat degradation.

BUT effective levels of natamycin remain significantly lower than

sorbate.

In 3 of the 5 yeasts tested, after a heat treatment of 70 oC for 10

minutes

300 ppm sorbate achieved 9 - 19 day shelf life,

5 ppm natamycin achieved 28 - > 50 day shelf life

Natamycin also achieved good control of Z. bailii - a strain known for

its resistance to traditional, chemical preservatives
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 Effect of temperature on natamycin retention
Danisco Specialities

Natamycin retention in orange juice, pH 3.5, at 25 °C [TJ020326].

22
20
Residual natamycin (ppm)

18 Natamycin addition levels:

16
14 5 ppm 10 ppm 20 ppm
12
10
8
6
4
2
0
0 5 10 15 20 25 30 35 40 45 50
Days incubation at 25 oC

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 Effect of temperature on natamycin retention
Danisco Specialities

Natamycin retention in orange juice, pH 3.5, at 8 °C [TJ020417]

20
18
16
Natamycin / ppm

14
12
10
8
6
4
2
0 10 20 30 40 50 60 70 80 90 100 110 120 130
Days at 8 oC

5 ppm 10 ppm 20 ppm

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 Natamax™ has better fungicidal activity than sorbate
Danisco Specialities

Comparison of cidal activity of natamycin and sorbate in orange juice

inoculated with Z. bailii and incubated at ambient temperature

Viable count results (CFU/ml)

Incubation time Natamycin Potassium sorbate
5 ppm 300 ppm
0 440 440

2.5 h 160 320

24 h < 10 640

3 days < 10 > 5 x 103

7 days < 10 > 5 x 103

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 Natamax™ has better fungicidal activity than sorbate
Danisco Specialities

Comparison of cidal activity of natamycin and sorbate in orange juice

inoculated with S. cerevisiae and incubated at ambient temperature

Viable count results (CFU/ml)

Incubation time Natamycin Potassium sorbate
5 ppm 300 ppm
0 640 640

2.5 h 450 490

24 h 10 280

3 days < 10 2000

7 days < 10 > 5 x 103

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 Natamycin: permitted addition levels
Danisco Specialities

South Africa

Fruit juice and pulp: 5 mg/kg natamycin

Natamycin has been used extensively for a long time in South Africa. This
country has always had wider authorisations for natamycin. South Africa was
where natamycin was first discovered.

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 Alicyclobacillus acidoterrestris – a fruit juice spoilage bacterium
Danisco Specialities

• Heat-resistant due to production of spores. Can survive typical heat

processes

• Acidophilic: grows at pH 2.5 to 6.0 at 25 to 60oC

• Causes flat-sour spoilage of fresh/pasteurised fruit juice

produces guaiacol - an off odour (medicinal/antiseptic smell)

• Nisin sensitive

5 IU/ml nisin controlled 104 cells or spores

after 7 d incubation at 37oC in lab media, pH 4

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 Nisaplin® can control growth of Alicyclobacillus acidoterrestris
Danisco Specialities

Minimum inhibitory concentration (IU/ml)

Apple juice Orange juice Grapefruit juice
Spores only

5 days at 25oC 5 5 5

5 days at 44oC 100 100 5

Spores plus
vegetative cells

5 days at 44oC 100 100 100

Data taken from Komitopoulou et al. 1999. Int. J. Food Sci & Technol 34: 81-85.
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 Nisin and heat effect on Alicyclobacillus acidoterrestris
Danisco Specialities

Addition of nisin increases heat sensitivity of spores

D va lu e in a p p le ju ic e , p H 3 .5
o
C

u n tre a te d s p o re s tre a te d
s p o re s w ith 5 0 IU /m l n is in

80 4 1 .1 5 2 3 .7 5

90 7 .3 8 4 .5 6

95 2 .3 0 1 .9 5

Data taken from Komitopoulou et al. 1999. Int. J. Food Sci & Technol 34:
81-85.
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 Natamax™: technical support from Danisco
Danisco Specialities

The technical support team offers customers

the following services free of charge:

• Analysis of juice for natamycin levels and

spoilage microflora

• Assistance and advice on Natamax™ levels

• Support for trials – including site visits and

product formulation

• Natamycin sensitivity testing

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 Nisaplin®: technical support from Danisco
Danisco Specialities

• Technical advice
• Support for trials – including site visits
and product formulation
• Analysis of juice samples
• Storage studies examining the retention
of Nisaplin® at different temperatures
• Analysis of spoilage microflora
• Nisin sensitivity testing

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