Note: Descriptions are shown in the official language in which they were submitted.
WO96109M2 8 8 PCT~S95/11956
Defatting Starch Containinq Waste Products
The invention relates to methods for at least the
functional removal of fatty substances from products which
consist for a major part of starch or starch-like products.
In particular, the invention relates to such methods for
removing residual fat from food products.
Food products for this invention are defined as any
fried, baked, pre-fried or pre-baked edible product which
contains both starch (or a starch derivative) and fatty
substances such as vegetable oils and fat.
lS Food products such as (pre-fried) French fries,
crisps, chips and other snack foods, cookies, cereals and
the like have to meet certain criteria as regards, for
example, size, colour and consistency in order to be
accepted by consumers. Products which do not comply with
set specifications after or during the manufacturing
process are either discarded as waste products or recovered
to be used in low economic value applications, such as feed
for cattle. It would be very useful if these waste or below
specification products could be used in applications having
a higher economic value.
The starch component of these products (which is
usually the major component) could be used as a
W096/09772 ; PCT~S95111956
- 2 - 22 00 ~ 88
carbohydrate source in fermentation processes such as for
the production of ethanol using yeast or other
micro-organisms or the fermentation production of enzymes.
However, the manufacturing process of (semimanufactured)
food products which are based on starch or starch-like
products often includes the use of fatty
substances, either as an ingredient or as a (pre-) frying
or baking medium.
When such waste products are employed to provide a
source of fermentable carbohydrate for fermentation
processes, it is found that fatty substances derived from
the starting waste product inhibit starch hydrolysis and
glucose fermentation when comparison is made to analogous
processes employing conventional starch products.
The present invention provides methods for processing
of products containing starch and/or starch derivatives and
also containing fatty substances wherein treatment is
carried out to functionally remove fatty substances. Within
the ambit of the present invention are, for example, novel
processes for improved use of waste products of the food
industry as carbohydrate sources for fermentations, e.g.
the fermentation production of ethanol. Such processes are
cost-effective and hence attractive.
In one aspect, the present invention thus provides a
method of processing a waste product containing as a major
component starch and/or one or more starch derivatives and
also containing fatty substances to provide a product of
W096/09772 2 2 0 0 5 8 8 PCT~S95/11956
higher economic value, said method including the step of
functionally removing fatty substances from said product or
a processing derivative thereof by lipase treatment.
The terminology "functionally removing" as used herein
will be understood to mean that residual products of the
enzymatic breakdown of fatty substances do not need to be
physically separated from the starch and/or starch
derivative(s) since they do not hamper the use of starch or
starch like products in many applications.
The applications of starch are many. Because starch
resulting from lipase treatment in a method of the
invention is relatively pure, it can be applied in most of
them. However, if starch of higher purity is required,
this can be easily achieved by separating residual
contaminants resulting from enzymatic breakdown of fatty
substances using known techniques.
Known applications of starch and starch-like products
in which products of methods of the present invention may
be employed include, but are not limited to, the food
industry (puddings, deserts, bakery products,
confectionery, soups, sauces, dressings, snacks, meat
products, beverages, milk products, jams and ice-cream), as
adhesives (on gummed paper, gummed tape, corrugated board,
laminating, bottle labelling, bags, wallpaper, carton
sealing, tube winding, etc.), in the paper industry
(internal sizing, surface sizing and surface coating), in
the textile industry (printing thickening, textile
W096/09772 ~ PCT~S95/119~
- 4 - ~ 20 0 5 8 ~
finishing and warp sizing) and many other miscellaneous
applications in the pharmaceutical industry, in building
materials and the like.
As hereinbefore indicated, methods of the present
invention are particularly preferred for providing starch-
containing products for use in preparation of fermentation
media, especially for use in providing a source of
fermentable carbohydrate for fermentation production of
ethanol. In this case, lipase treatment will be combined
with enzymic hydrolysis of starch and/or one or more starch
derivatives.
Lipases suitable for use in a method of the invention
may be obtained from bacteria, bacilli, yeasts, fungi or
higher eukaryotes. They may be modified chemically or by
recombinant techniques, they may be fusion proteins,
fragments of enzymes, etc. The only requirement that has
to be met is that the enzymatic activity must be present
and applicable in the process conditions. Particularly
suitable enzymes include the lipase Ml from Pseudomonas
pseudoalcaligenes (hereinafter lipase Ml), the S80000
lipase from Rhizopus arrhizus and lipase from Mucor miehei.
Many other suitable enzymes are known. They include but
are not limited to extracellular lipases, such as
triacylglycerol acylhydrolases, which are classified in the
EEC as E.C.3.1.1.3. These lipases are produced by many
(micro)organisms. Suitable microbial lipases have been
disclosed inter alia in U.S. Patent no.3,590,277. These
W096109M2 2 2 0 0 5 8 8 PCT~S95/11956
lipases were obtained from such diverse microorganisms as
Pseudomonas, Asperqillus, Pneumococcus, Sta~hylococcus,
Mycobacterium, Humicola. Mycotorula and Sclerotinia.
Lipases can be isolated from cultures of these organisms
and then used in the methods according to the invention.
The lipases can also be produced through recombinant DNA
techniques. Some lipase genes have already been cloned.
This enables higher production of lipases when
microorganisms harbouring these genes are cultured. It
also enables the production of more stable or more active
muteins of these lipases. All these enzymes can be applied
in the methods according to the invention.
In many cases, it may not be necessary to remove all
fatty substances to be removed by an enzymatic treatment.
When a high percentage of fat is present (in the order of
fifteen percent or more), it may be possible and
economically advantageous to remove the bulk of fatty
substances using another treatment such as a treatment with
hot water, e.g. having a temperature of 30-95~C, preferably
50-90~C. Such a pretreatment in combination with lipase
treatment as hereinbefore described constitutes another
embodiment of the invention.
In the exemplified application of the invention, hot
water treatment was not necessary. This is advantageous
because of the energy consumed and added costs incurred in
carrying out such a treatment.
Waste starch-containing food products are usually not
W096/09772 '~ PCT~S95/11956
- 6 - ~2 0058a
in a form in which they can be easily subjected to
enzymatic treatments. It will therefore usually be
necessary to convert such waste products to another form
prior to lipase treatment. Preferably, this is a suspension
in an aqueous medium. Preferred are aqueous suspensions
which contain about up to 50%, most preferably 10-20 %
solids (based on dry weight).
Methods according to the invention are preferably
applied to fried potato products, more particularly to
semimanufactured pre-fried potato products, of which French
fries are the most widely used example. French fries are
often sold (to consumers or to restaurants or snackbars and
the like) as semimanufactured pre-fried products. Such
pre-fried French fries contain about 10-20% of fatty
substances (usually of vegetable origin). Pre-fried French
fries which do not meet the desired specifications (e.g.
required food grade quality) will contain about the same
amount of fatty substances.
About 70~ of these fatty substances can be removed by
a pretreatment with hot water, after which the French fries
can be cut and suspended in an aqueous medium. As stated
before however, pretreatment with hot water may be omitted
altogether.
The resulting suspension can then be converted to a
source of fermentable carbohydrate by enzymic hydrolysis of
the starch present. This may be carried out at a
temperature of about 30-70OC and at a pH of about 4-6. The
W O 96/09772 ~ 2 0 U 5 ~ 8 PC~rrUS95/11956
optimum temperature and pH will, however, depend on the
enzyme or enzymes employed.
As shown by results presented below, in this way a
source of fermentable carbohydrate can be derived from pre-
fried French fries or an alternative starch-containing
waste product of the food industry which is far more
advantageous for ethanol production by virtue of the
decreased inhibition by fatty substances.
The amount in which enzymes have to be added in
processing methods of the invention will for a large part
depend on the enzyme applied. The person skilled in the
art knows how to determine the amount of enzyme needed.
Typically a lipolytic enzyme will be capable of about
2000-5000 conversions per second, which means that when in
a kilogram of raw product 10% fat (by weight) is present
with an average molecular weight of around 800 D, 2 pg of
an enzyme having a molecular weight of 30000 is necessary
to break down all the fatty substances in 1 hour. This is
of course only a rough estimate of what is possible under
ideal conditions.
The following examples illustrate the present
invention with reference to one fermenation process for
production of ethanol. It will be appreciated, however,
that starch-containing products prepared in accordance with
the present invention can be applied to many other
fermentation methods, including other fermentation methods
for production of ethanol from starch or starch-like
WO96109M2 PCT~S95111956
products well known in the art. 22~) 0 5 8 8
Examples
Methods
S Assay for the determination of lipase activit~
Activities of the lipase Ml and lipase of Mucor miehei
(Piccantase), expressed as ILUs, were determined on the
basis of hydrolysis of olive oil. The hydrolysis was
measured at 30~C in a pH-stat containing 10% olive oil in a
0.4 mM Tris buffer pH 9 in the presence of 20 mM sodium
chloride and l0 mM calcium chloride.
One ILU is defined as the amount of enzyme needed for
the release of one mmole fatty acid per minute under the
conditions of the test.
Extraction procedure
After incubation, the remaining triglycerides together
with the formed free fatty acids and diglycerides were
extracted from the incubation mixture (2 ml) with 5 ml of
hexane by rotating the glass tube for 30 minutes after
setting the pH at 2 with 4N HCl. The extraction was
terminated by centrifugation for 5 minutes at l000 rpm in a
table centrifuge to obtain a clear upper layer containing
triglycerides, diglycerides and free fatty acids.
220()~88
W096/09772 PCT~S9~/11
HPLC analYses
In the extraction solution, the residual amount of
triglyceride together with the amount of diglyceride and
free fatty acid formed were determined by HPLC.
Equipment and conditions
Pump: LKB (model 2150)
Detection: Refractive index monitor (Jobin Yvon)
Injection system: Perkin-Elmer ISS-101; lo ml
lo Integrator: Spectra Physics, Chromjet
Column: CP Microspher-Si (Chrompack),
lOO x 4.6 mm
Eluent: n-hexane/isopropylalcohol/formic
Acid: 975/25/2.5 (v/v), 1 ml/min
15 Temperature: ambient
Under the conditions described above, the ratio of the
refractive index responses between olive oil, oleic acid,
1,2 and 1,3-diacylglyceride were found to be 1.00, 0.98,
2.10 and 1.30, respectively, on the basis of peak height.
-
WO96109M2 ! PCT~S95/11956
- lO - 2200S~
EXAMPLE 1
Lipase Treatment of an Aqueous Suspension of Pre-Fried
French Fries
Pre-fried French fries (fried in vegetable oil) were
obtained in frozen form. After thawing, the fries were cut
into small pieces and a 20% (based on dry weight)
suspension was made in a lOOmM MES buffer pH 5.5 with a
Waring commercial blender.
Incubations were performed at 50~C over different time
periods and with different lipase dosages, as indicated in
Table l. The lipases used were the lipase Ml of
Pseudomonas pseudoalcaliqenes and lipase from Mucor-miehei.
Both lipases are products of Gist-Brocades and are
commercially available. After extraction of the remaining
triglycerides and hydrolysis products with hexane, these
products were analysed by HPLC as described hereinbefore.
The results are summarized in Table l.
W096/09772 22~ ~ 8 ~ PCT~S95/11956
-- 11 --
TABLE l
Percentage breakdown of vegetable oil from pre-fried French
fries upon lipase treatment.
s lipase dosage % breakdown after intli~t~d incub~tion time (hrs)
(ILU/ml) 0.5 1.0 2.0 4.0 16
-- 0 3 3 3 3 2
o Lipase Ml 2 5 8 12 13 41
" 4 7 9 12 19 49
" 10 10 14 24 32 44
" 20 10 17 30 49 60
" 40 13 25 41 58 63
Mucor miehei 2 4 4 5 9 9
" 4 4 5 6 10 10
" 10 4 5 6 8 12
" 20 6 8 10 18 12
" 40 5 6 8 12 16
From this table, it can be seen that the lipases used
showed their lipolytic properties on vegetable oil under
the conditions investigated. In comparison to the Mucor
miehei lipase, lipase Ml was found to be superior.
W096l~772 ~~ 2 PCT~Sg5Ulg~
- 12 -
EXAMPLE 2 ~
Ethanol Production by Fermentation Using Fermentable
Carbohydrate Derived From Pre-Fried French Fries
Pre-fried French fries were chopped into small pieces
with a blender. The dry solids content of the material was
determined by oven drying overnight at 70~C. Starch content
was measured using the Megazyme~ Starch Rit as
commercialized by Megazyme~. Enzyme dosages were determined
based on dry solids (33%) and starch content (75%).
Three 900 ml mash portions were set up in identical 11
bottles each containing an emulsion in water of 20% French
fries (based on dry weight), as prepared in a blender. The
emulsion was adjusted to pH 5.5 with 6N HCl and 6N NaOH as
required. Two bottles were treated with lipase Ml (500
grams/ton based on dry weight of French fries) for 4 and 16
hours in a 50~C waterbath. The third bottle was a control
without lipase pretreatment.
After lipase pretreatment, all three bottles were
adjusted to pH 6.5 with lN NaOH and transferred to a 60~C
waterbath and held for 30 minutes to equilibrate the
temperature prior to alpha amylase (Maxaliq~ ET) addition
in a dosage of 0.7 l/ton based on dry solids starch (ds
starch; 1 ton = 1000 kg). After mixing for 5 minutes, the
samples were autoclaved for 5 minutes at 107~C. Then the
bottles were returned to the waterbath, where they were
W09~09772 2 2 J U~ 8 8 PCT~S95/119~
'' t, ., ', .i _~
- 13 -
held at 93~C for an additional 2 hours. During this
incubation, the samples were stirred every 15 minutes.
After liquefaction, the samples were cooled to 600C,
adjusted to pH 4.8 and transferred to sterile 500 ml media
bottles. Fermentations were performed in triplicate.
Additional enzymes [Amigase~ (0. 8 l/ton ds starch),
Mycolase (0.07 l/ton ds starch), Maxazyme~ NPX (0.2 l/ton
ds starch)] were added and thoroughly mixed with the mash
prior to yeast addition. Fermiol was added at the
concentration of 0.2 kg/ton of mash. Penicillin was added
at the concentration of 500 units/ l of mash.
All nine media bottles were transferred for
fermentation over 63 hours to a shaker bath adjusted to
33~C/150 rpm. Samples were taken at 15, 24, 40,48 and 63
hours and analyzed by HPLC (Bio-Rad HPX-87H column, RI
detector) for total carbohydrates, fermentable
carbohydrates and ethanol.
HPLC results of ethanol production are shown in Table
2. Throughout the fermentation, the lipase treated French
fries produced a higher ethanol yield. Residual total and
fermentable carbohydrates are shown in Table 3. As expected
from the observed ethanol yields, there was a large
proportion of both fermentable and total carbohydrates in
the control sample at the end of fermentation. Ethanol
yields were calculated at 63 hours (grams ethanol
produced/grams of potential ethanol based on ds starch).
Control samples produced an average yield of 77%, compared
W096/09772 2 2 0 0 5 8 8 PCT~S95/~lg56
- 14 -
to an average yield of 90% and 92~ respectively for the 4
and 16 hour lipase pre-treated material.
This example shows clearly that lipase pre-treatment
of pre-fried French fries results in higher ethanol yields
in fermentation.
TABLE 2
Ethanol profile during a 63 hour fermentation
Time Ethanol produced (volume percent)
(hours) control lipase treated (4hrs) lipase treated (16 hrs)
O O. 00 O. 00 O. 00
3. 59 5. 80 5. 33
24 5. 84 7. 42 7. 70
6. 96 8. 38 8. 54
48 7.79 8. 92 9. 06
63 8. 29 9. 71 9.92
~20G~8~
W096/09772 ' PCT~S9S/119~6
TABLE 3
Residual total and fermentable carbohydrates.
5 sample residual carbohydrates after 63h (weight %)
total fermentable
Control 3.0120 2.1920
lipase treated (4hrs) 1.4600 0.6900
lo lipase treated (16hrs) 0.7614 0.0764
From this experiment another unexpected advantage becomes
clear. The amount of residual fermentable carbohydrates
was lower when a lipase-treated aqueous suspension of
French fries was employed. This will lead to a decreased
burden on the environment since the wasteload of the
reactor residue is lower.
