NL2019034B1 - Process for producing a cassava product - Google Patents

Abstract

The present invention relates to a process for producing a cassava product, more specifically to a process for producing a cassava cake or a cassava flour. The produced cassava product comprises a low level of cyanogenic glycosides. The invention further relates to a food product that comprises the produced cassava product.

Description

PROCESS FOR PRODUCING A CASSAVA PRODUCT

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a process for producing a cassava product, more specifically to a process for producing a cassava cake or a cassava flour. The produced cassava product comprises a low level of cyanogenic glycosides. The invention further relates to a food product that comprises the produced cassava product.

BACKGROUND OF THE INVENTION

Cassava (Manihot esculenta) is a woody shrub of the spurge family, Euphorbiaceae. It is extensively cultivated as an annual crop in tropical and subtropical regions for its edible starchy tuberous root. Cassava is the third largest source of food carbohydrates in the tropics, after rice and maize. It is one of the most drought-tolerant crops and is capable of growing on marginal soils.

When cassava roots are separated from the main plant, the roots undergo postliarvest deterioration. The roots, when damaged, normally respond with a healing mechanism within about 15 minutes. However, the same mechanism initiates after harvest and fails to switch off. It continues until the entire root is oxidized and blackened within two or three days after harvest, rendering it unpalatable and useless. The processing of cassava roots should therefore be initiated within the first 24 hours after harvest.

Cassava varieties are often categorized as either sweet or bitter, signifying a lower or higher presence of toxic levels of cyanogenic glycosides, respectively. The bitter cultivars are characterized by high levels of hydrogen cyanide (HCN) per kilogram of fresh roots. The sweet cassava root is preferably used for direct consumption as a staple food, while the bitter type is mostly processed into chips, pellets, starch and flour. Cassava flour is being used in many dietary applications, for example, as a substitute for other commercial flours in snack, bakery and pasta products. Cassava flour mainly comprises starch and fibers.

The contents of cyanogenic glycosides in the cassava root may vary, depending on cassava variety, harvest time, environmental conditions, and farm practices. Cassavas grown during drought are especially high in cyanogenic glycosides. Examples of cyanogenic glycosides in cassava roots are linamarin, lotaustralin and linustatin. Linamarin is the predominant cyanogenic glycoside in cassava plants. Cassava plants comprise endogenous linamarase enzymes. Linamarases hydrolyze the cyanogenic glycosides into cyanohydrin and a sugar group. Cyanohydrin spontaneously decomposes to HCN and acetone at temperatures higher than 30°C and pH higher than 4. The HCN then either dissolves readily in water or is released into the air.

It is critical during the processing of cassava roots, that the cyanogenic glycosides are removed, in order to reduce the residual cyanogenic glycosides to safe levels in the finished products. For the purpose of food applications, the cyanide content of the cassava product should be less than 10 mg of total HCN / kg of dry matter (food safety standards according to the WHO/FAO Codex Alimentarius Commission).

Improper preparation of cassava can leave enough residual cyanide to cause acute cyanide intoxication. Chronic, low-level cyanide exposure leads to the disease Konzo and is associated with the development of goiter and with tropical ataxic neuropathy, a nerve-damaging disorder that renders a person unsteady and uncoordinated.

With traditional processing techniques it is challenging to prepare cassava products with acceptable levels of cyanide. Both dry and wet traditional processing can only remove, under optimal processing conditions, a maximum of 90% of total HCN present in the cassava roots.

For example, in Nigeria it is allowed that garri, which is a cassava product, contains up to 20 ppm of cyanide, which is an indication that it is challenging to obtain garri products with levels of cyanide lower than 10 ppm. In 50 samples collected in Mozambique the average HCN in the samples was 43 mg of total HCN/kg flour. In an average year in Nampula, Province of Mozambique, only 14% of 119 flour samples had total cyanide contents of less than 10 ppm. Distribution curves of the total cyanide in flour show that the percentage of samples exceeding 100 ppm total cyanide increased from 6% in an average year to 43-65% in a low rainfall year in Nampula.

Dry processing is often used in African households. The cassava roots are washed and peeled and chopped in pieces. These pieces are laid out in the sun for the purpose of drying. After the drying in the sun, the dried pieces can be milled into a flour.

Brauman et. al. {Microbiological and biochemical characterization of cassava retting, a traditional lactic acidfermentation for Foo-Foo (cassava flour) production - 1996) describes an example of wet processing which applies fermentation of the cassava root to prevent the roots from rapid spoilage after harvest. Retting of cassava entails soaking root pieces in water for 3 to 4 days. During the consequent fermentation, roots are softened, the endogenous cyanogenic glycosides are reduced, and characteristic flavors are developed. The fermentation starts by the “contamination” with lactic acid bacteria which naturally occurs during the processing of the cassava roots.

Another wet processing method is the method to produce garri. First, the cassava tubers are peeled, washed and grated or crushed to produce a mash. This mash is placed in a porous bag and weights are placed on the bag for one to two days or more to press excess water and starch out. By day three, the cassava will lose a good amount of a water and become dry enough for the next step. It is then sieved and fried in an large clay frying pot with or without palm oil. The resulting dry granular garri can be stored for long periods. It may be pounded or ground to make a fine flour.

Sokari et al. (Changes in cassava toxicity during processing into gari and ijapu - two fermented food products, 1991) describes that traditionally processed cassava foods almost always contain residual cyanide. This led to the conclusion that traditional cassava processing was unlikely to remove all cyanide. It appeared that this might be due to insufficient activity of the endogenous linamarase. Efforts at supplementing the endogenous enzyme from exogenous sources either by inoculating cassava mash with linamarase preparations or with linamarase-producing microorganisms have shown, however, that very little additional detoxification could be achieved by adding more linamarase to the fermenting mash. WO 2012/003225 describes a process for producing a low cyanide cassava flour from bitter-type cassava roots. The process comprises providing a mash comprising crushed cassava root, adjusting a pH of the mash to a pH ranging from about 5.0 to about 7.5, incubating the mash at a temperature ranging from about 30°C to about 60°C for a time of at least 30 minutes, pressing the mash to remove excess water and provide a cassava cake, and processing the cassava cake to provide a low cyanide cassava flour having a crude fiber content ranging from about 1% to about 7% on a dry weight basis. It is described in WO 2012/003225 that it is advantageous to wash the cassava cake several times, which requires the presence of sufficient water supply. WO 2005/121183 describes a method for obtaining cassava flour from cassava roots, the method encompasses the following steps: grating the cassava roots, adding water to the grated matter to form a slurry, removing undesired components, such as proteins, among other components, from the slurry so as to obtain a product mass and, finally, drying the product mass. WO 2005/121183 further describes a device comprising means for performing the aforementioned method. The advantage of this device is that is provides a means for performing the aforementioned method in the immediate vicinity of the production location of the cassava roots.

Cassava starch is usually produced in factories in Asian countries, where several washing steps are applied, using large amounts of water, which is altematingly followed by water removal steps using large amounts of energy. The use of large amounts of water and several water removal steps leads to efficient removal of cyanogenic glycosides, because the cyanogenic glycosides, due to their solubility in water, are washed away, and if the cyanogenic glycosides are converted into HCN, the HCN evaporates during the several drying steps.

In Africa such factories are not yet available. Also there are additional challenges with the processing of the cassava roots. Electricity is not available in all areas and water availability is limited. Also, since the cassava roots need to be processed within the first 24 hours after harvest, and roads are of very poor quality, the transportation options are limited, thus even if such factories would be available, the harvested roots would not reach the factories in time.

Therefore there is a need for a method to produce a cassava product, such as cassava flour of cassava cake, which method can be applied using limited resources, while being suited for the processing of large quantities of cassava roots, while obtaining final cassava products with low levels of cyanogenic glycosides.

SUMMARY OF THE INVENTION

The inventors of the present invention have found a process for producing a cassava product that is low in cyanogenic glycosides and wherein the process can be suitably applied in areas with limited resources. The process can be suitably performed in a device as is described in WO 2005/121183. The method is further suited for the processing of large quantities of cassava roots.

More specifically the inventors have found a process for producing a cassava product comprising the steps of: a. providing peeled cassava roots comprising at least 30 mg cyanogenic glycosides per kg of dry matter; b. comminuting the peeled cassava roots to obtain a cassava slurry, wherein 100 parts by dry weight of cassava roots are combined with 50-400 parts by weight of water before, during and/or after the comminuting; c. adding linamarase to the cassava slurry in an amount of at least 4 units of linamarase activity per kg of cassava slurry to produce a linamarase containing cassava slurry; d. removing water from the linamarase containing cassava slurry to obtain a cassava cake with a water content of 30-70 wt.%; e. incubating the cassava cake to obtain an incubated cassava cake containing less than 10 mg of cyanogenic glycosides per kg of dry matter; and f optionally drying the cassava cake to produce a cassava flour.

The advantage of the present process is that the process does not require multiple washing steps and drying steps. A further advantage is that during the incubation step e) the incubated cassava slurry can be stored without temperature control and can be subsequently transported without temperature control and time pressure, i.e. the transportation does not need to take place within the first 24 hours after harvest.

Particularly advantageous of the present process is that it is suited for any type of cassava root and that it can handle a large variation in quality of cassava root input, without adapting the process. The cyanogenic glycosides concentration and thus the HCN level in the obtained cassava products is constant and well below food safety levels of 10 mg/kg of dry weight of the cassava product.

Without wishing to be bound by theory, it is believed that the present process is particularly efficient in removing cyanogenic glycosides due to the combination of process steps and the timing of the addition of the linamarases. Contrary to what the prior art states, the inventors have found that the addition of linamarase is effective, provided it is added before the pH of the cassava cake, due to fermentation, drops below a pH at which the linamarase does not work sufficient anymore.

The incubated cassava cake obtained by the present process can be used as such, or the incubated cassava cake can be dried to obtain a cassava flour comprising less than 15 wt.% water and less than 10 mg of cyanogenic glucosides per kg of cassava flour.

The invention further pertains to a food product comprising at least 1 wt.% of the cassava product obtainable by the process as described above.

DETAILED DESCRIPTION OF THE INVENTION A first aspect of the invention relates to a process for producing a cassava product comprising the steps of: a. providing peeled cassava roots comprising at least 30 mg cyanogenic glycosides per kg of dry matter; b. comminuting the peeled cassava roots to obtain a cassava slurry, wherein 100 parts by dry weight of cassava roots are combined with 50-400 parts by weight of water before, during and/or after the comminuting; c. adding linamarase to the cassava slurry in an amount of at least 4 units of linamarase activity per kg of cassava slurry to produce a linamarase containing cassava slurry; d. removing water from the linamarase containing cassava slurry to obtain a cassava cake with a water content of 30-70 wt.%; e. incubating the cassava cake to obtain an incubated cassava cake containing less than 10 mg of cyanogenic glycosides per kg of dry matter; and f. optionally drying the cassava cake to produce a cassava flour.

The term “cassava” as used herein refers to the roots of the plant Manihot esculenta.

The term “cyanogenic glycosides” as used herein refers to any molecule in which a sugar group is bonded through its anomeric carbon via a glycosidic bond to another group, which group comprises cyanide.

The term “comminuting” as used herein refers to the reduction of peeled cassava roots or parts thereof to small particles. Examples of comminuting are grinding, rasping, chopping or combinations thereof.

The term “linamarase” as used herein refers to enzymes that are capable of hydrolyzing cyanogenic glycosides, present in the cassava, into a cyanide comprising compound and a sugar group. Linamarase enzymes belong to the group of β-D-Glucosidases (EC 3.2.1.21), agroup of enzymes found in many plants and micro-organisms. Linamarase present in cassava has a good affinity (Km) for the cyanogenic glycosides found in cassava.

The term “unit of linamarase activity” is defined as the amount of enzyme that is capable of hydrolyzing 1.0 pmole of linamarin per minute at 25 °C, wherein the linamarin is present in an aqueous solution comprising around 250 ppm linamarin, 0.1 M phosphate buffer and a pH of 6.0. The linamarin breakdown is measured as the release of HCN. In comparative example 1 is described how this can be measured.

The term “fennentate” as used herein refers to the bulk growth of microorganisms in a growth medium. Suitable microorganisms are preferably selected from bacteria, yeasts, moulds and combinations thereof.

Cassava roots (step a)

The present invention is particularly suitable for processing peeled cassava roots comprising at least 50 mg cyanogenic glycosides per kg of dry matter, more preferably at least 100 mg and most preferably suitable for processing peeled cassava roots comprising between 150-2000 mg of cyanogenic glycosides per kg of dry matter.

The peeled cassava roots are preferably cleaned after peeling so that no dirt, stones, etc. are present on the peeled cassava roots.

Comminuting (step b)

It is preferred that the comminuting in step b) of the process of the present invention is performed by an industrial high-speed rasper. Comminuting of the cassava roots with an industrial high speed rasper leads to a >98% release of starch which is more than in any traditional way of cassava processing.

The comminuting is preferably performed such that a cassava slurry is obtained comprising fine cassava root pieces, more preferably that a cassava slurry is obtained comprising at least 90 wt.% of the cassava root pieces are smaller in length than 0.2 mm and most preferably that at least 90 wt.% of the cassava root pieces are smaller in length than 0.1 mm.

In step b) of the process of the present invention water can be added before comminuting; during comminuting; after comminuting; before and during comminuting; before and after comminuting; during and after comminuting; as well as before, during and after comminuting. It is preferred that the cassava roots are combined with water before and/or during the comminuting. More preferably the cassava roots are combined with water during the comminuting.

In one preferred embodiment it is preferred that 100 parts by dry weight of cassava roots are combined with 60-300 parts by weight of water, more preferably are combined with 70-200 parts by weight of water before, during or after the comminuting.

In another preferred embodiment it is preferred that the cassava slurry in step b) comprises between 5-15 wt.% of dry matter. When the cassava slurry comprises between 5-15 wt.% of dry matter, the cassava slurry is preferably sieved to reduce the fiber content to less than 2 wt.% by weight of dry matter of the cassava slurry.

Linamarase (step c)

Preferably linamarase is added to the cassava slurry in an amount of 4-40 units of linamarase activity per kg of cassava slurry to produce a linamarase containing cassava slurry, more preferably the linamarase is added to the cassava slurry in an amount of 8-35 units, most preferably the linamarase is added to the cassava slurry in an amount of 15-30 units of linamarase activity per kg of cassava slurry.

In a preferred embodiment the linamarase is added to the cassava slurry in an amount of at least 8 units of linamarase activity per kg dry matter of cassava slurry to produce a linamarase containing cassava slurry, more preferably the linamarase is added to the cassava slurry in an amount of 16-80 units, most preferably the linamarase is added to the cassava slurry in an amount of 30-60 units of linamarase activity per kg dry matter of cassava slurry.

In a preferred embodiment the linamarase is added in the form of an essentially pure enzyme or in the form of a linamarase source selected from: • cassava leaves extract; • a fermentate or extract thereof, comprising exogenous and/or endogenous linamarase.

In a more preferred embodiment the linamarase is added in the form of an essentially pure enzyme or in the form of cassava leave extract. In a most preferred embodiment the linamarase is added in the form of cassava leave extract.

Removal of water (step d)

Preferably in step d) of the process of the present invention, the water is removed from the linamarase containing cassava slurry to obtain a cassava cake with a water content of 35-65 wt.%, more preferably to obtain a cassava cake with a water content of 40-60 wt.%.

In a preferred embodiment step d) takes place within 4 minutes after completion of step c), more preferably step d) takes place within 2 minutes, most preferably within 1 minute after completion of step c).

It is further preferred that the combination of steps c) and d) take place within 10 minutes, more preferably within 5 minutes, most preferably within 2.5 minutes after completion of step b).

It is particularly preferred to fill the cassava cake in containers, such as bags, and to close these containers airtight. The volume of the containers preferably lies in the range of 20-500 L.

In a preferred embodiment the removed water may be re-used in the washing of the cassava roots before these roots are comminuted.

Incubation (step e)

Preferably the cassava cake is incubated at a temperature between 15-45°C for at least 5 hours to obtain an incubated cassava cake containing less than 10 mg of cyanogenic glycosides per kg of dry matter. More preferably the cassava cake is incubated at a temperature between 20-40°C for at least 5 hours to obtain an incubated cassava cake containing less than 10 mg of cyanogenic glycosides per kg of dry matter.

In case the outside temperature falls within in the temperature ranges mentioned in step e), it is preferred to incubate the cassava cake without temperature control. The incubation step can be suitably combined with transporting the cassava cake, especially if the incubation takes place in airtight sealed containers.

More preferably the cassava cake is incubated for at least 6 hours, even more preferably the cassava cake is incubated for 10-30 hours and most preferably the cassava cake is incubated for 15-24 hours.

The incubated cassava cake preferably contains less than 5 mg, more preferably less than 2.5 mg, and most preferably less than 1 mg of cyanogenic glycosides per kg of dry matter of the incubated cassava cake.

In the present process the amount of cyanogenic glycosides in the incubated cassava cake per kg of dry matter is preferably at least 2 times, more preferably at least 4 times, most preferably at least 10 times, lower than the amount of cyanogenic glycosides in the peeled cassava roots per kg of dry matter.

Lactic acid bacteria

In a preferred embodiment of the process of the present invention lactic acid bacteria are added during one or more of the process steps a) to d). The words “are added” also encompasses the advantageous contamination with lactic acid bacteria from the surroundings during the process. The lactic acid bacteria may be for example present on the outside of the cassava root before peeling.

In case of the addition of the lactic acid bacteria in the process of the present invention, the pH of the incubated cassava cake is preferably at least 1 pH unit lower than the pH of the cassava cake before incubation. More preferably the pH of the incubated cassava cake is at least 1.5 pH units, most preferably at least 2 pH units lower than the pH of the cassava cake before incubation.

In case of the addition of the lactic acid bacteria in the process of the present invention, the incubated cassava cake preferably comprises 0.2-2% by weight of dry matter of organic acids or salts thereof. These organic acids are preferably selected from lactic acid, acetic acid, and combinations thereof. Most preferably, the incubated cassava cake contains at least 0.2% lactic acid by weight of dry matter.

Drying step

It is preferred that the incubated cassava cake is dried at a temperature between 120-200°C to obtain a cassava flour comprising less than 15 wt.% water and less than 10 mg of cyanogenic glycosides per kg of cassava flour. More preferably the incubated cassava cake is dried at a temperature between 140-190°C and most preferably the incubated cassava cake is dried at a temperature between 160-180°C.

The obtained cassava flour more preferably comprises less than 14 wt.% of water, even more preferably less than 13 wt.% and most preferably less than 12 wt.% of water per kg of cassava flour.

The obtained cassava flour more preferably comprises less than 5 mg of cyanogenic glycosides, even more preferably less than 2.5 mg and most preferably less than 1 mg of cyanogenic glycosides per kg of cassava flour.

Preferably the drying of the incubated cassava cake is carried out in one or more drying devices selected from flash dryers, band dryers or drum dryers. More preferably the drying of the incubated cassava cake is carried out in a flash dryer.

The drying of the incubated cassava cake is preferably completed in less than 2 hours, more preferably in less than 1 hour and most preferably in less than 30 minutes.

In a preferred embodiment of the process according to the present invention, the obtained cassava flour is sieved to obtain a cassava flour with less than 2 wt.% of fibers by weight of dry matter. More preferably less than 1 wt.% of fibers and most preferably less than 0.5 wt.% of fibers by weight of dry matter.

The obtained cassava flour preferably comprises at least 95 wt.% of particles with an average particle diameter of less than 300 pm. More preferably the obtained cassava flour comprises at least 95 wt.% of particles with an average particle diameter of less than 250 μιη, most preferably less than 200 μιη. The particle diameter can be suitably determined by using a set of sieves with varying mesh sizes.

It is particularly preferred that the cassava product obtained by the present process is combined with one or more food ingredients to produce a food product.

Food product A second aspect of the invention relates to a food product comprising at least 1 wt.% of the cassava product obtainable by the process as described herein before. The embodiments as described herein before also apply to the food product of the present invention.

Preferably the food product comprises 5-90% by weight of dry matter of the cassava product obtainable by the process as described herein before. More preferably the food product comprises 10-80% by weight of dry matter and most preferably 15-70% by weight of dry matter of the cassava product obtainable by the process as described herein before.

The food product is preferably a food product selected from beer, bread, pastries, cakes, pasta, roux, bouillon and combinations thereof.

EXAMPLES

Cyanogenic glycoside measurement using LC-MS/MS

Sample preparation method for LC-MS/MS

For each analysis (in triplicate), 0.5 g sample was weighed into a 10 ml screw cap glass tube and spiked with 25 pL of 100 pg/ml internal standard (IS) phenyl-p-D-glucopyranoside stock solution. Then, 5 ml of 0.1 M HC1 were added and the mixture was vortexed and left to stand in the lab for five minutes. Then the mixture was vortexed again and centrifuged for 10 minutes at 3600 rpm. The supernatant was transferred into a 10 ml graduated glass tube and filled to the 5 ml mark with 0.1 M HC1. An aliquot of 0.5 ml was prepared by transferring 50 pL of the supernatant to a filter vial and adding 450 pL of water. The vial was vortexed and the filter pressed down.

Quantification was performed by external calibration on solvent standards. Solvent standards were prepared in water from linamarin, lotaustralin and IS stock solutions at concentrations in the range of 0.01-1.9 pg/ml of cyanogenic glycoside and 0.05 pg/ml ofIS. The internal standard (IS) was used to correct for the variation of the final volume and other possible errors made during the preparation. LC-MS/MS measurement

An LC-MS/MS system consisting of a liquid chromatograph ‘Waters Acquity UPLC I-class system’ equipped with a column heater, coupled to a ‘Waters Micromass Quattro Ultima Pt’ mass spectrometer with electrospray ionization (ESI) was used. All data were acquired and processed with Waters MassLynx 4.1 and QuanlynX software.

The analytical LC column was a Waters Xbridge 08 (3.0 x 150 mm, 5 pm particles) and the eluents were: A: milliQ water containing 5 mM ammonium formate, 0.1% formic acid (eluent A); and B: methanol/milliQ water 95/5 (v/v) containing 5 mM ammonium formate, 0.1% formic acid (eluent B).

The gradient program was as follows: - 0.0-6.0 min (5% B), - 6.0-8.5 min (50% B), - 8.5-10.0 min (100% B) and - 10.0-12.0 min (5% B).

The flow rate was set at 0.4 ml/min, the column temperature at 35°C and the injection volume at 10 pL. The MS operated in positive ESI mode and multiple reaction monitoring (MRM) was used for quantitation. The flow rates for the desolvation and cone gas were set at 559 L/h and 195 L/h, respectively. The source temperature was set at 120°C, desolvation temperature at 350°C, capillary voltage at 2.5 kV and cone voltage at 20 kV. Collision energies varied from 5 to 15 eV.

The transitions that were used for linamarin were: 265.3 (precursor ion) > 163.1 and 179.9 (product ions), and for lotaustralin were: 279.3 (precursor ion) > 163.1 and 179.9 (product ions). The collision energy was 10 eV for each transition and the cone voltage was 20 V.

Comparative Example 1

Cassava cake and cassava flour preparation

Cassava flour was prepared according to the method as described in example 1 of US 2012/0003356. Frozen cassava roots from Ghana were thawed, cleaned and peeled and roots were grinded with a blender to obtain a cassava pulp with a pH between 6.0-7.5. This cassava pulp was incubated at 40°C, 50°C or 60°C for 2 hours and subsequently excess liquid was removed by a pressing step and the total HCN content of this ‘pressed cake’ was determined. The remaining ‘pressed cake’ was oven dried for 24 hours at 55°C after which the total HCN content of this ‘dried flour’ was determined.

Samples of the resulting cassava pressed cakes and dried flours were analyzed for the total HCN content according to the method as described below.

Determination of total HCN content according to the Esser method

Total HCN refers to both bound HCN (e.g. in cyanogenic glycosides) and free HCN (e.g. volatile HCN or HCN present in cyanohydrin). For each analysis (in triplicate), 0.5 g of sample was weighed into a 50 ml screw cap tube. One of the tubes was used as a recovery sample by spiking with 915 pg of linamarin (20 mg/kg HCN equivalent). 50 ml of 0.25 M phosphate buffer pH 6 and 1 ml cassava leaf extract (serving as the enzyme solution to convert bound HCN into free HCN) was added each tube. The tubes were put away 18 hours into an oven set at 38°C, The tubes were put in ice water (approximately 30 min) and centrifuged at 3500 rpm during 10 min. A 0.5 ml of supernatant and 3.5 ml of buffer were transferred to a 10 ml screw cap tube. In the following order these color reagents were added: 0.2 ml of 0.18 M Chloramine T in milliQ and 0.8 ml of 0.23 M isonicotinic acid / 0.23 M 1,3-dimethylbarbituric acid. After 15 minutes, the absorbance was measured at 604 nm.

The calculation of the total HCN content was based on external calibration with HCN standards (in milliQ) prepared in the range of (0.04-0.2 pg/ml HCN equivalents).

Results

Table 1 shows the total HCN contents for both pressed cassava cake and the dried flour. The total HCN levels were all above 10 mg/kg dry weight in the dried cassava flour. The percentage of reduction after drying was between 8 - 84%.

Table 1

* This cassava root was another type of cassava root compared to the roots that were placed at 40°C and 60°C.

Example 1

The kinetics of cyanogenic glycosides in caished cassava slurry with and without linamarase was assessed.

Enzyme preparation

Young leaves from cassava plants were used for the enzyme extraction. 500 gram leaves were grinded with a kitchen blender in 1500 ml 0.1M phosphate buffer pH 6.0. The obtained slurry is sieved over a nylon filter cloth with average pore size of 250 pm. The obtained filtrate was heated in a water bath at 45°C during 4 hours to release free HCN. The filtrate was filtered over a Whatman Grade 1573 filter paper to remove residual debris. The linamarase activity of the leave extract isolate was quantified according to the method below.

Linamarase activity measurement

The quantification of the linamarase activity was based on the release of HCN from linamarin. The assay medium included 0.5 ml of enzyme extract and 4.5 ml 0.1 M phosphate buffer pH 6.0 containing a known quantity linamarin. Samples were taken every 30 minutes over a period of 4 hours and analyzed for free HCN (according to the method as described in comparative example 1, but only measuring free HCN). Linamarase activity was calculated from the slope of the straight line showing the increase HCN increase during the first 4 hours. One unit of

linamarase activity was expressed as the amount of enzyme that will hydrolyze 1.0 pmole of linamarin (measured as release of HCN) per minute at pH 6.0 at 25 °C.

Preparation of cassava slurry

Frozen cassava roots from Ghana were thawed, cleaned and peeled and subsequently these roots were grinded with a kitchen blender to obtain a cassava slurry with a pH between 6.0-7.5. The cassava slurry was divided in two: cassava slurry A and B. 35 units of linamarase activity was added per kg of cassava slurry B. As a linamarase source the leave extract as described herein before was used. Both cassava slurries were incubated at 30°C.

Samples

Samples were taken every hour during the first 8 hours and one sample was taken after 24 hours. For the cassava slurry B also samples every 10 minutes were taken during the first 4 hours. The breakdown of cyanogenic glycosides were measured in these samples using LC-MS/MS (as is described herein before). The advantage of using this LC-MS/MS technique is that is allows for a very accurate measurement of the amount of the different cyanogenic glycosides present in the samples.

Results

The initial breakdown rate of cyanogenic glycosides in cassava slurry A was approximately 50 mg/hr/kg slurry and in cassava slurry B the rate was about 600 mg/hr/kg slurry.

In cassava slurry B the total amount of cyanogenic glycosides (linustatin, linamarin, lotaustralin and neolinustatin) was hydrolyzed within the first 2 hours after processing. While for cassava slurry A even after 24 hours there were still cyanogenic glycosides remaining at a concentration of 20 mg/kg of dry weight of the cassava slurry. The HCN in these cyanogenic glycosides is bound and will therefore remain in the dried cassava product.

Example 2

Fresh cassava tubers from Jangamo region, Inhambane provence, Mozambique containing approximately 20-70 ppm total HCN / kg fresh weight are processed using an AMPU device as described in WO 2005/121183. Approximately 4000 kg roots/hr are washed to remove soil and peeled to remove the outer skin of the roots. Peeled roots are chopped into small pieces of approximately 2-4 cm and transported to the GEA Hovex HDR300 high speed rasper and simultaneously approximately 2000 litre/hr clean water is added.

The 6000 litre/ hour cassava slurry from the rasper has a pH between 5.5 and 6.5 and temperature between 20-34°C and is subsequently sent to an AlfaLaval STNX912 decanter to remove excess fruit water and to produce a cassava cake of approx. 45% solids. Additional linamarase enzyme extracted from cassava leaves (prepared as described in example 1) can be added in line directly after the rasper, and is mixed homogenously in the cassava slurry before the water removal step by the decanter.

The pressed cassava cake from the decanter is in-line packed into 21 kg plastic bags sealed and stored at ambient temperature for at least 1 day.

Samples from the pressed cassava cake produced with and without additional enzyme were taken after the 1 day storage and the levels of cyanogenic glycosides (bound HCN) in the samples is measured (according to the method as described in comparative example 1). In the cassava cake with additional enzymes the amount of cyanogenic glycosides is below 10 mg of cyanogenic glycosides per kg of dry matter. Whereas the in the cassava cake without additional enzymes the amount of cyanogenic glycosides is higher than 10 mg of cyanogenic glycosides per kg of dry matter.

After the storage, the cassava cake comprising about 55% moisture is dried to obtain a cassava flour, by dispersing the cassava cake into a hot air stream of max. 160°C generated by a gas fueled or electric heater. The obtained cassava flour is transported along the dryer conduct and terminated in a bank of cyclones, which remove the dried cassava flour from the air stream. All volatile components such as free HCN are stripped and removed via the air stream, but residual cyanogenic glycosides will remain in the final product.

Claims (15)

A process for manufacturing a cassava product, the process comprising the steps of; - providing peeled cassava roots comprising at least 30 mg of cyanogenic glycosides per kg of dry matter; - pulverizing the peeled cassava roots to obtain a cassava slurry, wherein 100 parts by weight of cassava roots, on a dry weight basis, are combined with 50-400 parts by weight of water, during and / or after pulverizing; - adding linamarase to the cassava slurry in an amount of at least 4 units of linamarase activity per kg of cassava slurry to obtain a linamarase-containing cassava slurry; removing water from the linamarase-containing cassava slurry to obtain a cassava cake with a water content of 30-70 wt. %; incubating the cassava cake to obtain a incubated cassava cake containing less than 10 mg of cyanogenic glycosides per kg of dry matter; and - optionally drying the cassava cake to obtain a cassava flour. The process of claim 1, wherein the incubated cassava cake is dried at a temperature of between 120-200 ° C to obtain a cassava flour containing less than 15% by weight of water and less than 10 mg of cyanogenic glycosides per kg. contains cassava flour. The process according to claim 1 or 2, wherein the amount of cyanogenic glycosides in the incubated cassava flour per kg of dry matter is at least 2 times lower than the amount of cyanogenic glycosides in the peeled cassava roots per kg of dry matter. The process according to any of claims 1-3, wherein lactic acid bacteria are added during one or more of the process steps a) to d). The process of claim 4, wherein the pH of the incubated cassava cake is at least 1 pH unit lower than the pH of the cassava cake prior to incubation. The process according to any of claims 1-5, wherein the combination of steps c) and d) takes place within 10 minutes after the completion of step b). The process according to any of claims 1-6, wherein in step b) the cassava roots are combined with water before and / or during pulverization, preferably during pulverization. The process according to any of claims 1-7, wherein the linamarase is added in the form of a substantially pure enzyme or in the form of a linamarase source, which is selected from: - an extract of cassava leaves; - a fermentate or an extract thereof, containing exogenous and / or endogenous linamarase. The process according to any of claims 1-8, wherein the drying is completed in less than 2 hours. The process according to any of claims 1-9, wherein the cassava slurry in step b) comprises between 5-15% by weight of dry matter. The process of claim 10, wherein the cassava slurry is sieved to reduce the fiber content to less than 2% by weight based on the dry matter weight of the cassava slurry. The process according to any of claims 1-9, wherein the cassava flour is sieved to obtain a cassava flour with less than 2% fiber by weight based on the dry matter weight. The process according to any of claims 1-12, wherein the cassava product is combined with one or more food ingredients to obtain a foodstuff. A foodstuff comprising at least 1% by weight of the cassava product obtainable by the process of any one of claims 1-13. The foodstuff according to claim 14, wherein the foodstuff is selected from beer, bread, pastries, cake, pasta, roux, broth and combinations thereof.