Technology and processing (Module 3)
Overview on aims and the objectives in the 5th project year (June 2009 - May 2010)
In order to enable production of healthier cereal foods, new ingredients, new processing tools, and knowledge on process-induced changes on cereal matrix is needed. Therefore, the work in this module included development of dry milling and wet fractionation processes, as well as development of new enzymes, enzymatic processing and fermentation in order to produce foods with increased levels of grain aleurone components and structural features delivering good sensory properties.
The objectives in this final project period were to
- analyse a set of commercial wheat brans to be compared to new fractions produced
- provide other partners with new well characterized bran fractions
- develop new tools for development and control of grain fractionation processes (markers, understanding of cohesion/adhesion)
- develop processing diagrams for whole grain debranning and milling
- develop diagrams for dry cereal bran fractionation at tissue and sub-cellular scale for production of healthy ingredients
- develop bioprocesses to impact the health profile of cereal constituents in vitro and in situ
- develop enzyme and fermentation assisted wet processing technologies for isolation of selected functional bioactive compounds
- study xylanases as preferred tools for production of arabinoxylan derived carbohydrates
- elucidate the impact of bioprocessing on the structural and compositional aspects of bran and bread with a focus on phenolic compounds
- extract phenolic compounds from wheat and rye bran
- use enzyme technology for production of arabino-oligosaccharides in situ
- develop innovative bioprocessing technologies for incorporation of whole grain and bran-derived ingredients in cereal foods
- reveal mechanisms important for retaining sensory quality or bread.
- elucidate the role of cereal phytochemicals in end product quality
- use high pressure technology for improvement of quality of gluten free bread
- produce HealthFlour and whole grain flour for consumer end product evaluation
- compare ultra fine milling and turbo air classification with cryogenic ultra fine milling and electrostatic separation
- produce aleurone from bran for application trials
- assess the economics of cryo/ambient separation and electrostatic/turbo air classification.
Work performed and the main achievements
In Module 3, different bran materials have been collected, analysed and distributed to partners. In the application of fluorescence properties to determine the histological composition of isolated cereal fractions, in the last year, the scientists managed to get excellebt fluorescence imaging. The work showed that wheat tissue cohesion may likely be estimated via laser-induced breakdown spectroscopy experiments. A micromechanical device was used to carry out peel tests with simultaneous optical observations. Under well specified conditions, the device could be used to evaluate the intertissue adherence in different locations of the surface of the grain using different dissected samples. The peel force increased with the intensity of oxidative pretreatment of cereal tissue interfaces composed of feruloylated arabinoxylans.
Different milling technologies allow bran grinding at ambient (impact milling with a screen) and cryogenic (counter rotating pin mill) conditions. High throughputs can be obtained and the milling conditions determine the size distribution of the different tissues and components and their separability.
Electrostatic separation and turbo air classification are both good technologies to produce an aleurone enriched fraction. A very fine particle size device is indispensable to reach the goal of collecting separately the walls and cytoplasm of aleurone cells. As ambient milling technology provides a good range of granulation sizes, the more expensive cryogenic grinding technology could be avoided.
Aleurone products are a good option for fibre enrichment in bakery (bread, biscuits, froze dough) and provide an alternative to wheat bran. They have the benefits of a dough behaviour similar to that of a reference. The production cost of the electrostatic positively charged fractions, rich in aleurone layers, is in line with that of other products rich in micronutrients.
The work on xylanase enzymes revealed that the flexibility of the thumb region in a partcular glycoside hydrolase family 11 enzyme studied is higher than previously proposed, and that alteration of key thumb residues influences the binding of the substrate as well as the release of product from the active site. The thumb region and its dynamics, therefore, are of vital importance for the specific catalytic action of xylanases. Combined with the information that is available on the inhibition sensitivity, functional stability and substrate selectivity of BsXynA, this knowledge provides prospects for targeted engineering of the xylanase to improve its performance in cerealbased applications.
Hydrolytic enzymes with ferulic acid esterase activity had the most significant effect on the extractability of ferulic acid from wheat bran materials. In breadmaking trials, the combined treatment of wheat bran with enzymes and yeast resulted in the best baking behaviour in terms of bread volume and crumb softness. Enzymes and yeast improved the quality of bran enriched bread also when added in the dough mixing phase. However, long and effective bioprocessing is needed to release ferulic acid from bran. In addition, yeast fermentation is required to increase the folate content of bran and bread and to keep the material microbiologically safe.
A xylanase enzyme able to in situ enrich bread in arabinoxylan oligosaccharides without compromising dough and final bread quality was identified.
In a French breadmaking process, several cereal outer layer fractions appeared very promising (at 0.8% fnal ash content) for the production of appealing and nutritionally enriched breads, especially fractions rich in aleurone material obtained from electrostatic separation. At 1.5% final ash content (equivalent to a wholemeal bread), the breads were more penalized, but some remained very attractive in term of flavours.
In a semi-industrial bread production trial, HealthFlour showed the same behaviour as the whole wheat flour. Both HealthFlour and whole wheat breads had similar crumb mechanical features (hardness and resilience).
Reactive compounds in the bran decrease gluten’s ability to aggregate, and cause the negative effects on breadmaking. The accessibility of the reactive compounds to interact with the gluten seems to be of key importance. Thus, not only the bran’s composition, but also its fine structure should be regarded to better understand the effects of bran.
Sensory analysis showed that consumers in different parts of Europe have different preferences for wholegrain wheat bread. Finnish consumers prefer most the dark and less soft wheat breads with intense flavour, probably due to the typical high consumption of rye and other dark and flavour-intense breads in Finland. A wholegrain claim had positive impact on consumer preferences in Belgium. Belgian consumers seemed to be more influenced by the claims than Finnish consumers.
The work on breads was also complemented with work on ready to eat cereals. Processing steps and parameters were worked out for producing tasty ready to eat cereals enriched with bran.
Finally, sourdough technology was used for the production of gluten free oat bread. Addition of sourdough yielded breads with greater specific loaf volume, a more open crumb structure and even distribution of gas cells. The increase in hardness during the storage period (staling) was overall only minimal.
In order to enable production of healthier cereal foods, new
ingredients, new processing tools, and knowledge on
process-induced changes on cereal matrix is needed. Therefore the work
in this module includes development of
dry milling and wet fractionation processes, as well as development of
new enzymes, enzymatic processing and
fermentation in order to produce foods with increased levels of grain
aleurone components and structural features
delivering good sensory properties.
The objectives were:
In Module 3, the provision of cereal batches and production of flours with selected extraction rates for different project partners was continued. Partners have developed research tools for the understanding and control of fractionation processes on whole grain and demonstrated the production of functionally and nutritionally improved HEALTHGRAIN FOOD-CT-2005-514008 13 (178).
whole grain flours as well as of wheat and rye bran fine fractionation for ingredient production, using in particular cryogenic and electrostatic technologies. Ultrafine grinding at ambient temperature allowed a better dissociation of the different bran tissues than cryogenic grinding. Youna Hemery was the first in the Healthgrain project to defend her thesis, entitled ”Fractionnement par voie sèche du son de blé (Triticum aestivum L.) pour l'obtention d'ingrédients alimentaires: des bases de la fractionnabilité aux propriétés fonctionnelles des fractions‘.
Bioprocesses selectively impacting the health profile of major and minor cereal constituents both in vitro and in situ were developed as were enzyme and fermentation assisted wet processing technologies for isolation of selected functional bioactive compounds. To this end, previously developed xylanases were produced, site directed mutagenesis of a family 11 xylanase was performed and heterologous expression of a family 10 xylanase in Bacillus subtilis was executed. For a number of enzymes tested on different bran materials, the yield of extraction of potentially health promoting arabinoxylan oligosaccharides was negatively correlated with the arabinose to xylose ratio of the wheat bran arabinoxylan. Ball milling was an alternative way for production of wheat and rye bran derived arabinoxylan oligosaccharides. The particle size of the wheat bran material was dramatically reduced in the ball-milling processing, and the cell wall structures of bran were no longer microscopically detectable.
Enzymatic hydrolysis of dry milling fractions of wheat in combination with yeast fermentation was studied with the aim to liberate phenolic compounds from the cell wall matrix. Enzyme treatment with ferulic acid esterase activity was the most important factor in releasing ferulic acid from the bran cell wall matrix, while yeast fermentation balanced the microbial quality of samples.
As far as innovative bioprocessing technologies for incorporation of whole grain and bran-derived ingredients in breadmaking goes, in situ enrichment of bread in arabinoxylan oligosaccharides was possible using at least one thermophilic xylanase, but high levels of high molecular weight soluble arabinoxylan could not be obtained. As the level of arabinoxylan oligosaccharides that can theoretically be obtained in white wheat flour based bread is probably insufficient for physiological effects, enrichment of breads with arabinoxylan rich fractions or bran was used for the production of arabinoxylan oligosaccharides rich bread. Three different enriched dietary fibre fractions (wheat bran, rye bran and aleurone) were evaluated as supplements in the production of arabinoxylan oligosaccharides in breads. Peeling of grain before flour preparation and addition of xylanase in breadmaking significantly improved the sensory quality of wholemeal wheat breads. Also, naturally occurring levels of alkyl resorcinols did not affect the leavening properties of wheat and rye bran breads. Alkylresorcinols were not degraded during the baking process.
With regard to pilot scale and demonstration activities, bread sticks, wholegrain flour, barley and wholegrain pasta have been distributed to Module 4 for a long term nutrition study.
The industrial feasibility study of ‘Healthflour’ production revealed that ‘Healthflour’ has less potential food safety issues than whole wheat flour. The production cost of ‘Healthflour’ is about 4% higher than the production cost of regular whole wheat flour.
In order to enable production of healthier cereal foods, new
ingredients, new processing tools, and knowledge on process-induced
changes on cereal matrix is needed. Therefore the work in this module
includes development of dry milling and wet fractionation processes, as
well as development of new enzymes, enzymatic processing and
fermentation in order to produce foods with increased levels of grain
aleurone components and structural features delivering good sensory
properties.
The objectives in the third project year were:
- to develop biomarkers for identification of the histological composition of cereal fractions
- to further develop dry milling using cryogenic techniques and electrostatic separation to obtain bran and flour with new composition
- to develop extremophilic xylanases and starch modifying enzymes and use them in production of oligosaccharides and resistant starch
- to study the use of fermentation as a means to control digestibility
- too produce prototype products for the intervention studies in Module 4
- to evaluate the feasibility of different technologies being developed
The work in Module 3 is organised in 5 work packages (WP):
WP 3.1
Supply of cereals and cereal fractions
Flour and bran fractions were prepared and distributed for the large
scale experiments. A selection of commercial wheat brans was collected
to be analysed as a reference for the ingredients developed in the
project and those already available commercially.
WP 3.2
Novel technologies for whole grain dry fractionation
Tools were developed for the understanding and control of the cereal
fractionation processes, whole grain
fractionation to produce functionally and nutritionally improved
(pseudo-)whole grain flours, and bran fine fractionation using cryogenic
and electrostatic technologies. Specific recommendations have been
proposed to quantify the relative proportions of wheat tissues into
technological fractions from unknown wheat cultivars batches using
marker technologies. The development of equipment for rapid
determination of tissue has suffered some delay.
The laser ablation technique allows accessing the native grain tissue
mechanical properties. Whole grain
fractionation efforts focused on debranning/milling combinations for
production of flours alternative to whole grain flours. Use of cutinase
was shown to degrade the cutin layer adjacent to aleurone, but the
impact on subsequent milling remains to be demonstrated.
WP 3.3
Novel wet processing technologies for production of bioactive
compounds or fractions
Xylanases suited for the production of soluble (prebiotic) arabinoxylan
(oligosaccharides) were developed. Addition of amylose-lipid complexes
to starch gels resulted in gels with significantly higher resistant
starch content, showing their possible health improving potential. It
was recognized that bran fermentation has positive effects on the baking
quality, and drying technology was developed to allow for production of
stable modified brans.
WP 3.4
Novel technologies to produce nutritionally optimised food from
cereal grains and insight in process
induced changes in bio-active compounds
Progress was made in applications of both enzyme and fermentation
technology in baking. A new psychrotolerant xylanase had a strong bread
improving effect, especially when using fibre sources rich in particular
arabinoxylan. Fermentation improved structure and released free
phenolic acids in breads very rich in outer grain layers and grain
fibre. Use of laccase improved the volume and bread crumb of 100% oat
breads suitable for coealic patients.
WP 3.5
Pilot/industrial/demonstration activities
The Healthflour concept was brought to larger scale trials. Several
wheat milling fractions were tested in pasta making and large scale
baking experiments. Bread and biscuit prototypes with high content of
whole grain or grain outer layers, or with retarded glycemic response
were developed and produced in quantities needed for intervention
trials. The samples were supplied to long term nutrition studies. The
consortium has finalized flow charts of the most innovative processes.
The processes were ranked for their innovativeness, technological issues
and marketing potential.
As a prerequisite to novel processing routes for cereals, markers to
determine the tissue composition during grain fractionation were
developed. In addition, work was carried out on novel dry and wet
processing techniques as well as fermentation technologies, all of these
offering perspectives for routes increasing the health potential of
cereals and their fractions. Factors influencing dough and bread
structure were analysed to be able to optimise processes with respect to
sensory quality and starch digestibility
The objectives for the second project year were:
- to develop a prototype equipment for determination of the histological composition of cereal fractions
- to develop dry milling using cryogenic techniques and electrostatic separation to obtain bran and flour with new composition
- to develop fermentation as a way to increase bioactivity and palatability of brans and breads containing grain outer layers
- to analyse factors influencing dough and bread structure to be able to optimise processes with respect to sensory quality and starch digestibility
- to start demonstration activities evaluating the feasibility of the technologies developed on pilot and industrial scale
The work in Module 3 is organised in 5 work packages (WP):
WP 3.1
Supply of cereals and cereal fractions
Based on a preliminary analysis of the processing quality of 8 samples
from different market offers of the wheat cultivar Tiger, a necessary
amount of Tiger grain of German origin has been purchased, analysed in
more detailed and milled into fractions requested by the various
partners.
WP 3.2
Novel technologies for whole grain dry fractionation
The research on molecular markers as a tool to assess tissue
concentrations in different cereal fractions focused on
a novel method to measure relative wheat germ contents and on rapid
estimation of alkyl resorcinols. Wheat germ agglutinin was chosen as a
marker for wheat germ tissues and an immunoenzymatic ELISA assay for
their quantitative estimation is under further development. A
colorimetric assay for alkylresorcinol estimations holds promise for
implementation but still needs to be validated. FT-IR methodology
calibrated with tissues of known compositions will be complementary to
biochemical marker technology to identify and quantify tissue
compositions. The development of equipment for rapid determination based
on fluorescence has suffered from insufficient performance of some
optical components. The work on the development of a grain fractionation
diagram to produce low-pericarp-low-crease whole grain flour
("Healthflour") should result in an alternative to the "Whole Grain
concept". This technology appears relatively simple to implement with
combinations of already widely accessible debranning and milling
equiments. Pre-treatment of the grains is also an important step of
grain processing and can profoundly affect the grain behaviour upon
processing. A prerequisite for successful development in this area is a
better knowledge on the nature and energy of the linkages between
tissues or inside tissues. To this aim, impulsional laser ablation has
been introduced as a tool. The exploration of the cryogenic milling of
bran to get particles much below the aleurone cell size is on the way.
Model studies have shown that in cryogenic milling, the grinding of bran
is more efficient.
WP 3.3
Novel wet processing technologies for production of bioactive
compounds or fractions
During the reporting period, several new enzymes were developed for
specific purposes. Penicillium xylanases were engineered for better
activity and altered substrate specificity. Thermophilic xylanases were
produced for conversion of the arabinoxylan in bread into
oligosaccharides with prebiotic potential, without affecting their
technological contribution to the process. The xylanases from P.
haloplanktis and B. subtilis were the most promising enzymes for
releasing arabinoxylan and oligosaccharides from cereal fractions.
Arabinoxylan oligosaccharides were produced on small (g) to large scale
(kg) for trials in Module 4. Work on starch modifying enzymes showed
that selected enzymes can contribute to gel strength. Similar
observations were made for starches in the presence of amylose lipid
complexes. When applied to products, both strategies may lead to slower
digestion of the starch in the gastro-intestinal tract. The potential of
enzymatic pre-treatments to improve the milling behaviour of wheat
kernels and the nutritional properties of the obtained milling streams
was demonstrated. The use of xylanases during conditioning lowers energy
input requirements during milling, and increases contents of soluble
fibre in the resultant flour. Enzymatic peeling of partially debranned
kernels through the use of Xylanase rich preparations opens the door for
selective removal of tissues which give the bran undesired sensory
properties. Fermentation technology increased the availability of
bio-active components and of soluble fibre in bran and enhanced its
technological quality in the production of cereal products. Both yeast
and spontaneous fermentation were tested in absence and presence of
added enzymes. Yeast fermentation of peeled bran increased the contents
of soluble arabinoxylan, folates and free ferulic acid and delivered
bran with very good technological and sensorial quality when used in
bread making. Work in a last subworkpackage focussed on phenolic
components and yielded an optimised procedure for isolating
alkylresorcinols from different cereals. This procedure was used for
obtaining nearly gram quantities of alkylresorcinols from wheat, durum
wheat and rye.
WP 3.4
Novel technologies to produce nutritionally optimised food from
cereal grains and insight in process induced changes in bio-active
compounds
The major items studied were the interference and processability of
arabinoxylan in wheat breadmaking, and reduction of starch digestibility
of wheat breads while maintaining the sensory quality. A large number
of grain fractions both from dry and wet milling in WP 3.2. and 3.3.
were studied, and numerous samples were delivered to Module 4 for
studying of in vitro and in vivo digestibility. Product prototypes were
developed to be evaluated in Module 4 as candidates for the long term
intervention studies. Enzyme technology and fermentation were the major
technological tools developed, and were successfully applied also to
gluten free flours, such as buckwheat and rice, to improve their
bread-making potential.
The extractability, structure and physical properties of arabinoxylan,
the major constituent of wheat and rye dietary fibre, have a range of
influences on the texture and also gastrointestinal functions of cereal
foods. Arabinoxylans were shown to interfere with gluten formation both
directly by interactions with gluten, and indirectly by affecting water
distribution. Use of solubilizing xylanase, alone and in combination
with cross-linking enzymes, increased the levels of extractable
arabinoxylan, resulting in improved dough strength, bread volume and
crumb softness. As short-chain arabinoxylo-oligosaccharides are
candidate prebiotics, various xylanases were compared for their
production in situ. In order to produce physiologically relevant levels
of arabino-oligosaccharides, ingredients with high starting levels of
arabinoxylan are needed. Transglutaminase proved useful to promote
network formation in baking of gluten-free flours, and significantly
decreased chewiness and hardness of buckwheat and rice based breads.
Lactobacillus plantarum was identified as useful starter culture for
improving the shelf life of gluten-free breads. It also significantly
improved the mechanical properties of the breads.
Bread prototypes containing aleurone particles and whole and cut kernels
and good sensory perception of texture were developed for Module 4. A
new way of pre-cooking kernels prior to baking was developed in order to
make a bread with 80% of whole or cut kernels. Recipes were developed
for bread containing 25% aleurone and 6-8 % dietary fibre, as well as
for whole grain bread and biscuits. Preliminary studies for use of durum
wheat aleurone in pasta were started.
WP 3.5
Pilot/industrial/demonstration activities
Two type of wheat based breakfast cereals were produced. One type was
enriched with high purity aleurone fraction and one control sample
contained pure wheat starch, gluten and fibre.
Ring shaped wheat based breakfast cereals were produced using a Buhler
twin screw extruder and a fluid bed dryer. Breakfast cereals designed to
contain 0 or 9 g of aleurone per portion were produced. The basic
recipe consisted of wheat flour, aleurone fraction, water and salt for
one product. For a second product, 5% of sugar was added. For the
control product, the composition of aleurone was reconstituted with pure
wheat starch and gluten and with a commercial fibre. These samples were
shared with different partners from Module 4. The sweetened samples
were preferred over the unsweetened and will be chosen for larger scale
production of portions for the long term study. Furthermore, a list
(matrix) of technologies and applications of specific wheat fractions or
ingredients was prepared to assess the technologies and applications
with highest potential to be up-scaled to industrially produce a healthy
wheat product.
Overview on aims and the objectives in the 1st project year (June 2005 - May 2006)
The overall objective of the work in Module 3 is to develop
technologies and processing methods for nutritionally optimised cereal
foods and new food ingredients from whole grains.
Generally stated, the technology and processing module focuses on the
development of novel dry and wet
technologies to isolate and process new fractions of high nutritional
impact from grains and milling byproducts. Technologies are developed
to concentrate health relevant components or to incorporate them in
consumer foods in the form of new ingredients. The work deliberately
focuses on enzyme and fermentation technologies to bring about good
eating quality in combination with good nutritional properties. In this
context, it is important that product texture, an important factor both
to sensory perception and bio-availability of phytochemicals and
starch, will be optimised. In addition, research will also be devoted
to the development of gluten free food products.
The corresponding technical objectives for the whole programme read as follows:
- to develop new food ingredients of high nutritional impact by isolation and/or processing of new cereal fractions using economically viable technologies
- to develop cereal foods of high nutritional impact and gluten free products using economically viable technologies
- to study process-induced changes of biomarkers and bioactive compounds in European grains
- to evaluate and demonstrate the feasibility of the developed technologies in industrial processing The objectives for the first reporting period were the following:
- to provide for cereal batches and flours with selected extraction rates for project partners
- to develop novel technologies for whole grain and bran fractionation
- to develop a method for determination of tissue composition in grain fractions based on molecular markers
- to develop enzyme technology to selectively modify the health profile of cereal constituents in vitro and in situ
- to develop enzyme assisted wet processing technologies for isolation of selected functional bioactive compounds or production of new fractions of high nutritional impact from cereals and cereal sidestreams
- to develop fermentation assisted wet processing technologies for production of new fractions from cereals and cereal sidestreams
- to develop bioprocessing technologies for incorporation of whole grains and novel ingredients in cereal foods
- to understand the role of gluten and arabinoxylan in structure formation of products high in bran
