WO2005060727A2

WO2005060727A2 - Improvements to decontamination of seeds

Info

Publication number: WO2005060727A2
Application number: PCT/GB2004/005286
Authority: WO
Inventors: Keith Warriner; William M. Waites
Original assignee: The University Of Nottingham
Priority date: 2003-12-24
Filing date: 2004-12-17
Publication date: 2005-07-07
Also published as: GB0329929D0; WO2005060727A3

Abstract

The present invention relates to a method of reducing contamination of seeds and sprouted seeds. The method comprises treating the seeds with a decontamination agent. The decontamination agent may be a compound that is activated in use, by an acid generated in situ, to generate chlorine dioxide. The decontamination agent may be Ozone gas or an other suitable agent including hydrogen peroxide. The method may also include subjecting the seeds to a heat treatment step.

Description

IMPROVEMENTS TO DECONTAMINATION OF SEEDS

The present invention relates to methods of reducing contamination of seeds and sprouts cultivated from seeds from human pathogens such as E. coli and Listeria mono cy to genes.

The consumption of sprouts cultivated from seeds is now very popular but has been associated with numerous outbreaks of food borne illness. Sprouts cultivated from seeds are cultivated at a temperature (in the range of 25-35°C) and a humidity (greater than 80%) optimal for their sprouting and growth. However, such temperatures and humidities provide an ideal environment for breeding of various micro-organisms, including human pathogens.

Various methods have been considered for reducing contamination of seeds and sprouts cultivated from seeds but care must be taken to avoid harsh treatments that prevent the seeds from subsequently sprouting or that leave the seeds or sprouts cultivated from seeds with an unnatural taste or in a state not suitable for human consumption. For example, heat treatment sufficient to destroy pathogens can prevent germination of the seeds.

Furthermore sterile seeds should ideally not be produced as post-harvest contamination could take place in the absence of any competitive microflora. The presence of competitive microflora is critical as they act as a biological buffer to inhibit the growth of pathogens during sprouting. The microflora compete to use the nutrients present thus limiting the nutrients available for pathogen growth. Accordingly, there still exists a need for useful methods for reducing contamination of seeds and sprouts cultivated from seeds that will be eaten that address the aforementioned problems, are practical, and cost effective. In the context of the present invention the phrase "sprouted seeds " includes sprouted seeds such as beansprouts and other sprouts cultivated from seed and eaten. Examples of the types of seeds that require decontamination are mung beans, alfalfa seeds and cress seeds.

Accordingly in a first aspect the present invention provides a method of reducing contamination of seeds and sprouted seeds, the method comprising treating the seeds with a compound that is activated in use, by an acid generated in situ, to generate chlorine dioxide.

The compound activated in use to generate chlorine dioxide preferably comprises sodium chlorite or sodium chlorate. The compound may be provided in the form of a solution or a solid. The compound may be a stable sodium chlorite solution. Most preferably the compound is a stabilised chlorine dioxide (SCD) solution consisting of a highly refined blend of oxy-chloro species containing sodium chlorite. A suitable SCD solution is available as Harvest Wash supplied by Vernagene Ltd.

The compound is activated by acid to generate anti-microbial chlorine dioxide. The acid is generated in situ, for example, by bacterial activity during their growth on nutrients released from the germinating seeds . Generation of the acid in situ by the bacterial activity during the germination of the seeds means that the compound is stable before use and therefore readily handled. Furthermore, the release of chlorine dioxide is localised around the bacterial activity and is therefore slower and prolonged compared to the activation by the addition of acid, other than that produced in situ by bacterial activity, which produces a high concentration of chlorine dioxide but this is only present for a short period of time. It is believed that when sodium chlorite is added to the seed steep solution the metabolism of endogenous microflora produces organic acids that activate the chlorite in the micro-environment thereby generating localised chlorine dioxide production. When sodium chlorate is used it must first be reduced to sodium chlorite by the enzyme nitrate reductase, which is expressed in coliforms including E. coli. Chlorine dioxide can then be generated as described above.

The seeds are preferably soaked in a solution of the compound for 12 to 36 hours, preferably 18 to 30 hours and most preferably 24 hours.

Where the acid is generated in situ by bacterial activity during the germination of the seeds the compound activated in use to generate chlorine dioxide is preferably present in amounts of from at least lOOppm to at least 500ppm or more, a preferred amount is at least lOOppm, and a most preferred amount is 125ppm.

According to a second aspect the present invention provides a method of reducing contamination of seeds and sprouted seeds, the method comprising the steps, in any order, of i) subjecting the seeds to a heat treatment; and ii) treating the seeds with a decontamination reagent.

The heat treatment step is preferably carried out before treatment with the decontamination reagent. The heat treatment step preferably comprises submerging the seeds in water at a temperature of from 95 to 100°C, preferably at boiling point, for a period of less than 20 seconds, preferably 5 to 15 seconds, most preferably 10 to 15 seconds, for example 10 seconds. Preferably, the heat treatment step is followed by quenching of the seeds in a liquid at a temperature of 0 to 6°C, preferably 4°C or less. The liquid is advantageously a solution of the decontamination reagent. In this embodiment the decontamination reagent is used to quench the seeds after treatment with hot water. This quenching process is advantageous as it rapidly cools the seeds and increases uptake of the decontamination reagent.

Treatment with a decontamination reagent can be carried out by one of the following processes:

Process One - The decontamination reagent may be a compound activated in use, by an acid generated in situ, to generate chlorine dioxide.

The compound is activated by an acid, which is generated in situ, for example, by bacterial activity during their growth on nutrients released from the germinating seeds. Generation of the acid in situ by the bacterial activity during the germination of the seeds means that the compound is stable before use and therefore readily handled. Furthermore, the release of chlorine dioxide is localised around the bacterial activity and is therefore slower and prolonged compared to the activation by the addition of acid, other than that produced in situ by bacterial activity, which produces a high concentration of chlorine dioxide but this is only present for a short period of time.

It is believed that when sodium chlorite is added to the seed steep solution the metabolism of endogenous microflora produces organic acids that activate the chlorite in the micro-environment thereby generating localised chlorine dioxide production. When sodium chlorate is used it must first be reduced to sodium chlorite by the enzyme nitrate reductase, which is expressed in conforms including E. coli. Chlorine dioxide can then be generated as described above.

Where the acid is generated in situ by bacterial activity during the germination of the seeds the compound activated in use to generate chlorine dioxide is preferably present in an amount of from at least lOOppm to at least 500ppm or more, a preferred amount is at least lOOppm, most preferably 125ppm.

The seeds are preferably soaked in a solution of the compound for 12 to 36 hours, preferably 24 hours.

Process two - The decontamination reagent may be a compound activated prior to or in use to generate chlorine dioxide.

The compound activated prior to or in use to generate chlorine dioxide preferably comprises sodium chlorite. The compound may be provided in the form of a solution or a solid. The compound may be a stable sodium chlorite solution. Most preferably the compound is a stabilised chlorine dioxide (SCD) solution consisting of a highly refined blend of oxy-chloro species containing sodium chlorite. A suitable SCD solution is available as Harvest Wash supplied by Vernagene Ltd. The compound is preferably activated by an acid. The acid may be added to the compound and may, for example, be phosphoric acid or citric acid.

When the acid is added to the solution of the compound the acid should be present in amounts of from 3 to 10% w/v or 3 to 10 % v/v. The compound activated prior to or in use to generate chlorine dioxide is preferably present in the solution in an amount of from at least 25ppm to at least 250ppm or more, a preferred amount is at least 25ppm and a most preferred amount is 50ppm.

The seeds are soaked in a solution of the compound and acid, added to activate the compound, for a period of from 2 to 10 minutes, preferably 3 to 7 minutes and most preferably 5 minutes.

The compound activated in use to generate chlorine dioxide may alternatively comprise a stable chlorine dioxide preparation, which can be activated by spraying. A suitable stable chlorine dioxide preparation is available as Harvest Wash TC supplied by Vernagene Ltd. In this embodiment the seeds or sprouted seeds are sprayed with the compound activated in use to produce chlorine dioxide.

Process three - Ozone gas as a decontamination reagent.

The ozone gas is preferably present in air contained in a treatment chamber in an amount of from at least 30ppm to at least 50ppm or more. A most preferred ozone gas content is 30ppm.

In further processes it is envisaged that other suitable decontamination reagents including hydrogen peroxide, peracetic acid, ethanol, cetylpyridium chloride or organic acid vapour, for example acetic acid vapour could be used. Also provided are seeds and sprouted seeds having reduced levels of human pathogens. Further provided are seeds and sprouted seeds obtainable by the methods of the present invention.

A number of embodiments of the invention will now be described by means of the following examples.

Examples

In the examples reference to "non- activated Harvest Wash" refers to Harvest Wash activated in use by acids generated in situ by bacteria.

Reference to "activated Harvest Wash" refers to Harvest Wash activated before use or in use by addition of an acid, e.g. phosphoric acid.

TVC provides an indication of microflora naturally present in sprouted seeds and how this microflora is affected by the methods of the present invention.

Protocol for mung bean inoculation and sprouting

Batches of mung beans (600g) were submerged in maximum recovery diluent containing bioluminescent E. coli and L. monocytogenes (10⁷ colony forming units/ml (cfu/ml)) for 30 mins. The beans were then dried on sterile filter paper for 2 hours at room temperature and separated into lOOg batches. The batches were then treated with a decontamination reagent for 5 mins. The mung beans were then submerged in sterile distilled water at 30 °C for 24 hours. The water was removed and sprouting continued for 3 days at 30°C. The beans were watered daily using sterile distilled water. The sprouting mung beans were harvested after 4 days and the weights of the bean sprouts was recorded and lOg batches were removed for microbiological analysis.

Experimental procedures for bacterial enumeration

A) TVC was carried out by plating on tryptic soya agar and incubating at 30°C for 48 hours.

B) Escherichia coli was enumerated on tryptic soya agar containing 30μg/ml of kanamycin and incubating at 37°C for 24 hours.

C) Listeria monocytogenes was enumerated on Listeria selective agar (Oxford formula) and incubating at 30°C for 48 hours.

Comparative Example

Decontamination of mung beans with Harvest Wash activated with phosphoric acid

The inoculated mung beans were soaked in a Harvest Wash solution (500ml activated prior to use with phosphoric acid) and left for 5 minutes. The decontaminant solution was replaced with sterile water and mung beans germinated.

Table 1 : Bacterial counts derived from mung bean seeds decontaminated by Harvest Wash activated prior to use with phosphoric acid.

TVC is Total Viable Count. Initial Load is bacterial count of inoculated seed before treatment. ND: Denotes "not detected". 0: Denotes mung beans treated in sterile distilled water in the absence of any decontamination reagent.

The results set out in Table 1 show that levels of lOOppm Harvest Wash, or greater, activated with phosphoric acid reduced the levels of E. coli and L. monocytogenes on inoculated mung beans to below the level of detection. Table 2: Bacterial counts derived from the resultant sprouts of the mung beans decontaminated by Harvest Wash activated prior to use with phosphoric acid.

TVC is Total Viable Count. *: Denotes concentration of Harvest Wash activated with phosphoric acid used to decontaminate the beans. ND: Denotes "not detected" . 0: Denotes mung beans treated in sterile distilled water in the absence of decontamination reagent.

L. monocytogenes was not detected on bean sprouts derived from mung beans decontaminated with Harvest Wash activated with phosphoric acid. E. coli levels in bean sprouts were reduced as higher levels of activated Harvest Wash were used but were not reduced to levels that could not be detected. The comparative example therefore shows that the use of Harvest Wash activated with phosphoric acid, although reducing all human pathogens to undetectable levels on the mung bean seeds, does not prevent human pathogens from breeding during germination of the seeds and therefore being present on the sprouted seeds. Examples illustrative of the present invention

1. Decontamination of seeds using hot water pasteurisation and Harvest Wash activated with phosphoric acid

The inoculated mung beans were submerged in one litre of boiling water for 10-15 seconds. The beans were then rapidly transferred to cold (4°C) activated Harvest Wash solution (500ml activated prior to use with phosphoric acid) and left for 5 minutes. The decontaminant solution was replaced with sterile water and mung beans germinated over four days at 30°C. Counts were performed on the subsequent sprouts.

Table 3: Bacterial counts derived from sprouts of inoculated mung beans decontaminated by a combination of hot water and Harvest Wash activated prior to use with phosphoric acid.

TVC is Total Viable Count. *: Denotes concentration of Harvest Wash activated with phosphoric acid used to decontaminate the beans. Initial load: Denotes counts recovered from inoculated beans prior to treatment. ND: Denotes "not detected". 0: Denotes mung beans treated in sterile distilled water in the absence of a decontamination regent.

In comparison with the Comparative Example the use of hot water treatment with Harvest Wash activated with phosphoric acid significantly reduced numbers of E. coli on sprouts and E. coli were not detected when levels of 50ppm or above Harvest Wash activated with phosphoric acid were used in combination with a hot water treatment. L. monocytogenes were not detected on the sprouts when levels of 25ppm or above Harvest Wash activated with phosphoric acid were used in combination with a hot water treatment. 2. Ozone decontamination of mung beans

Inoculated mung beans were submerged in boiling water for 15 seconds prior to placing in a chamber containing air into which ozone gas was introduced. The ozone gas concentration was maintained constant in the chamber throughout a 5 minute treatment period.

Table 4: Bacterial counts derived from sprouts of mung beans treated with hot water and different ozone gas concentrations.

TVC is Total Viable Count. *: Denotes concentration of ozone gas with which the mung beans were treated. 0: Denotes mung beans treated in sterile distilled water in the absence of a decontamination regent. ND: Denotes "not detected".

Treatment of inoculated beans with an initial hot water treatment and ozone gas resulted in those beans treated with at least 30ppm ozone having no E. coli or L. monocytogenes detected on the resultant sprouts. TVC levels remain fairly constant. 3. Non-activated Harvest Wash

Part A - Without heat treatment The beans were initially soaked in water containing non-activated Harvest Wash for 24 hours at 30°C. The Harvest Wash solution was then replaced by sterile water and the mung beans sprouting continued for a further 3 days. Counts were then carried out on the sprouts.

Table 5: Bacterial counts derived from sprouts of mung beans soaked in non-activated Harvest Wash.

TVC is Total Viable Count. NT: Denotes "not treated". ND: Denotes "not detected". HW: Denotes Harvest Wash.

Treating mung beans with non-activated Harvest Wash is more effective than treating with activated Harvest Wash as seen from comparison with the Comparative Example. Part B - With heat treatment The beans were initially submerged in 1 litre of boiling water for 10 seconds before being soaked in water containing non-activated Harvest Wash for 24 hours at 30 °C. The Harvest Wash solution was then replaced by sterile water and the mung beans sprouting continued for a further 3 days. Counts were then carried out on the sprouts.

Table 6: Bacterial counts derived from sprouts of mung beans soaked in non-activated Harvest Wash after an initial hot water treatment.

TVC is Total Viable Count. NT: Denotes "not treated" . HT: Denotes 10 second submersion in boiling water. ND: Denotes "not detected". HW: Denotes Harvest Wash. Example 3 shows that non-activated Harvest Wash at levels of at least lOOppm, with or without hot water treatment, provides consistent inactivation of E. coli and L. monocytogenes during mung bean sprouting.

Furthermore use of non-activated Harvest Wash at levels of at least lOOppm enables the initial hot water treatment to be omitted.

TVC levels remain fairly high and constant with non-activated Harvest Wash used with or without initial hot water treatment, providing a good biological buffer system.

Table .7: Optimisation of non-activated Harvest Wash mung bean treatment: - bacterial counts derived from sprouts of mung beans soaked in non- activated Harvest Wash without heat treatment.

NT: Denotes "not treated" . ND: Denotes "not detected" *: Denotes concentration of non-activated Harvest Wash used to decontaminate the beans. Non-activated Harvest Wash at concentrations of at least lOOppm is sufficient to suppress the growth of E. coli and L. monocytogenes during mung bean sprouting.

TVC levels remain fairly high and constant, providing a good biological buffer system.

5. Sodium Chlorite

The beans were soaked in distilled water containing a decontamination reagent for 24 hours at 30° C. The decontamination reagent containing solution was then replaced by sterile water and the mung beans sprouting continued for a further 3 days. Counts were then carried out on the sprouts.

Table 8: Bacterial counts derived from bean sprouts of mung beans treated with non-activated Harvest Wash or sodium chlorite without heat treatment.

NT: Denotes "not treated" .

ND: Denotes "not detected" .

* Denotes concentration of decontamination reagent used to decontaminate the beans. Example 5 shows that sodium chlorite is equally as effective as Harvest Wash at inactivating inoculated E. coli and L. monocytogenes.

6. Yields of Bean Sprouts from treated Mung Beans

Table 8a: Treatment with Hot Water and Ozone Gas.

Table 8b: Treatment with Non-activated Harvest Wash.

7. Decontamination of Alfalfa seeds

Alfalfa seeds were inoculated by submersion in a cocktail of E. coli and L. monocytogenes (at a concentration of 10⁷ cfu/g) for 20 mins. Seeds were dried at room temperature for 24h prior to use. Batches (20g) were decontaminated by soaking in distilled water containing a decontamination reagent for 24 hours at 30°C. The decontamination reagent containing solution was then replaced by sterile water and the alfalfa seed sprouting continued for a further 4 days at 30°C. Counts were then carried out on the sprouts.

Table 9: Bacterial counts derived from sprouts of decontaminated alfalfa seeds.

NT: Denotes "not treated" .

ND: Denotes "not detected"

* Denotes concentration of decontamination reagent used to decontaminate the beans.

When considering Alfalfa seeds both sodium chlorite and non-activated Harvest Wash reduce levels of E. coli and L. monocytogenes to levels that cannot be detected. Furthermore sodium chlorite is equally as effective as Harvest Wash at inactivating inoculated E. coli and L. monocytogenes .

Claims

1. A method of reducing contamination of seeds and sprouted seeds, the method comprising treating the seeds with a compound that is activated in use, by an acid generated in situ, to generate chlorine dioxide.

2. A method of reducing contamination of seeds and sprouted seeds, the method comprising the steps, in any order, of iii) subjecting the seeds to a heat treatment; and iv) treating the seeds with a decontamination reagent.

3. The method of Claim 2 wherein the decontamination reagent is a compound activated in use, by an acid generated in situ, to generate chlorine dioxide.

4. The method of Claim 1 or Claim 3 wherein the compound activated in use to generate chlorine dioxide comprises sodium chlorite or sodium chlorate.

5. The method of any of Claims 1, 3 or 4 wherein the compound activated in use to generate chlorine dioxide is provided in the form of a solution or a solid.

6. The method of any of Claims 1 or 3 to 5 wherein the compound activated in use to generate chlorine dioxide is a stable sodium chlorite solution.

7. The method of any of Claims 1 or 3 to 5 wherein the compound activated in use to generate chlorine dioxide is a stabilised chlorine dioxide (SCD) solution consisting of a highly refined blend of oxy-chloro species containing sodium chlorite.

8. The method of any of Claims 1 or 3 to 7 wherein the compound is activated by acid generated in situ by bacterial activity during their growth on nutrients released from the germinating seeds.

9. The method of any of Claims 1 or 3 to 8 wherein the seeds are soaked in a solution of the compound for 12 to 36 hours.

10. The method of any of Claims 1 or 3 to 9 wherein the seeds are soaked in a solution of the compound for 18 to 30 hours.

11. The method of any of Claims 1 or 3 to 10 wherein the seeds are soaked in a solution of the compound for 24 hours.

12. The method of any of Claims 1 or 3 to 11 wherein the compound activated in use to generate chlorine dioxide is present in amounts of from at least lOOppm to at least 500ppm or more.

13. The method of any of Claims 1 or 3 to 12 wherein the compound activated in use to generate chlorine dioxide is present in an amount of 125ppm.

14. The method of Claim 2 wherein the decontamination reagent is a compound activated prior to or in use to generate chlorine dioxide.

15. The method of Claim 14 wherein the compound activated prior to or in use to generate chlorine dioxide comprises sodium chlorite.

16. The method of Claim 14 or 15 wherein the compound activated prior to or in use to generate chlorine dioxide is provided in the form of a solution or a solid.

17. The method of any of Claims 14 to 16 wherein the compound activated prior to or in use to generate chlorine dioxide is a stable sodium chlorite solution.

18. The method of any of Claims 14 to 16 wherein the compound activated prior to or in use to generate chlorine dioxide is a stabilised chlorine dioxide (SCD) solution consisting of a highly refined blend of oxy-chloro species containing sodium chlorite.

19. The method of any of Claims 14 to 18 wherein the compound activated prior to or in use to generate chlorine dioxide is activated by an acid.

20. The method of Claim 19 wherein the acid added to the solution of the compound activated prior to or in use to generate chlorine dioxide is present in amounts of from 3 to 10% w/v or 3 to 10 % v/v.

21. The method of any of Claims 14 to 20 wherein the compound activated prior to or in use to generate chlorine dioxide is present in the solution in an amount of from at least 25ppm to at least 250ppm or more.

22. The method of any of Claims 14 to 21 wherein the compound activated prior to or in use to generate chlorine dioxide is present in the solution in an amount of 50ppm.

23. The method of any of Claims 14 to 22 wherein the seeds are soaked in a solution of the compound and acid, added to activate the compound, for a period of from 2 to 10 minutes.

24. The method of Claim 2 wherein the decontamination reagent is Ozone gas.

25. The method of Claim 24 wherein the Ozone gas is present in air contained in a treatment chamber in an amount of from at least 30ppm to at least 50ppm or more.

26. The method of Claim 24 or 25 wherein the Ozone gas is present in air contained in a treatment chamber in an amount of 30ppm.

27. The method of Claim 2 wherein the decontamination reagent includes hydrogen peroxide, peracetic acid, ethanol, cetylpyridium chloride or organic acid vapour.

28. The method of any of Claims 2 or 3 to 27, when dependent on Claim 2, wherein the heat treatment step is carried out before treatment with the decontamination reagent.

29. The method of any of Claims 2 or 3 to 28, when dependent on Claim 2, wherein the heat treatment step comprises submerging the seeds in water at a temperature of from 95 to 100 °C for a period of less than 20 seconds.

30. The method of claim 29 wherein the water is at boiling point.

31. The method of any of claims 2 or 3 to 30, when dependent on Claim 2, wherein the heat treatment step is followed by quenching of the seeds in a liquid at a temperature of 0 to 6° C.

32. The method of claim 31 wherein the liquid is a solution of the decontamination reagent.

33. Seeds and sprouted seeds having reduced levels of human pathogens.

34. Seeds and sprouted seeds obtainable by the methods of the present invention.

35. A method of reducing contamination of seeds and sprouted seeds substantially as described herein and with reference to the Examples.

36. Seeds and sprouted seeds substantially as described herein and with reference to the Examples.