Multi-enzyme product with glucoamylase, proteolytic and...

Details Multi-enzyme product with glucoamylase, proteolytic and... Multi-enzyme product with glucoamylase, proteolytic and...

2001-07-24

2003-12-23

Hendricks, Keith (Department: 1761)

Food or edible material: processes, compositions, and products

Fermentation processes

Of farinaceous cereal or cereal material

C426S060000, C435S175000

Reexamination Certificate

active

06667066

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to a multi-enzyme product with glucoamylase, proteolytic and xylanase activities and a method for producing same by solid state fermentation of wheat bran with
Aspergillus niger.
BACKGROUND OF THE INVENTION
It is known to produce ethanol from corn starch by an enzymatic method comprising a stage for liquefying the starch with an alpha-amylase for hydrolyzing the starch to dextrins, and then a saccharification stage by a glucoamylase (also called amyloglucosidase) for hydrolyzing the dextrins to glucose, and finally a stage for fermenting the latter to ethanol.
The use of alpha-amylase and glucoamylase enzymes is generally satisfactory when relatively pure starch milk, obtained by the wet milling of corn, is used as starting material, but when it is desired to substitute wheat starches or wheat flours for corn starch, no satisfactory results are obtained with these two enzymes alone because of the presence of hemicelluloses, which increase the viscosity of the saccharified flour worts to the extent that this creates a problem for carrying out the method. It is necessary to use, at the saccharification stage, auxiliary enzymes such as cellulases and hemicellulases in order to reduce the viscosity and remedy this problem. Moreover, it is desirable also to use proteases during saccharification so as to hydrolyze the proteins in the flour and thus enrich the wort with soluble nitrogen in anticipation of the subsequent alcoholic fermentation stage. The traditional supply of nitrogen source necessary for the growth of yeasts during this fermentation may thus be reduced.
All these enzymes are individually commercially available in purified form, but have the disadvantage of being relatively expensive and therefore of increasing the cost of producing ethanol from wheat. In addition, compositions have to be formulated from individual enzymes, which complicates the method.
A need therefore exists for an inexpensive multi-enzyme product combining glucoamylase, proteolytic and hemicellulase activities to produce ethanol from wheat flours at a low cost.
The invention aims to satisfy this need.
SUMMARY OF THE INVENTION
The present invention relates to a multi-enzyme product exhibiting glucoamylase, proteolytic and xylanase activities, characterized in that it consists of wheat bran fermented with an
Aspergillus niger
strain, said enzymatic glucoamylase, proteolytic and xylanase activities being present at the following minimum values:
glucoamylase: at least 100 GU per gram of dry matter,
proteolytic: at least 100 PU per gram of dry matter,
xylanase: at least 100 XU per gram of dry matter, provided that at least one of the following conditions is satisfied:
the glucoamylase activity is at least 750 GU per gram of dry matter
the xylanase activity is at least 300 XU per gram of dry matter.
The glucoamylase activity is preferably at least 1 500 GU per gram of dry matter and/or the xylanase activity is at least 400 XU per gram of dry matter.
Also, the proteolytic activity is preferably at least 400 PU per gram of dry matter.
The invention also relates to a method of producing this multi-enzyme product, characterized in that it comprises the stages consisting in (a) taking wheat bran; (b) moistening and heat-treating said bran so as to pasteurize it or sterilize it; (c) inoculating the resulting wheat bran with an
Aspergillus niger
strain; (d) the bran being in the form of a layer at least 10 cm thick, fermenting it in the solid state in a reactor which is aerated and stirred periodically for a period of 1 to 3 days, at a temperature of 28-38° C., preferably 32 to 36° C., said bran being adjusted to an initial moisture content of 50 to 60 wt. % which is substantially maintained during the duration of the fermentation, under aeration conditions appropriate for avoiding accumulation of carbon dioxide which is harmful to the fermentation in the reactor and a rise in temperature due to fermentation above the recommended range, until the fermentation product exhibits the following minimum enzyme activity values:
glucoamylase: at least 100 GU per gram of dry matter,
proteolytic: at least 100 PU per gram of dry matter,
xylanase: at least 100 XU per gram of dry matter, provided that at least one of the following conditions is satisfied:
the glucoamylase activity is at least 750 GU per gram of dry matter
the xylanase activity is at least 300 XU per gram of dry matter.
The glucoamylase activity is preferably at least 1 500 GU per gram of dry matter and/or the xylanase activity is at least 400 XU per gram of dry matter.
Also, the proteolytic activity is preferably at least 400 PU per gram of dry matter.
The
Aspergillus niger
strain is preferably chosen from the NRRL 3112 strain, the ATCC 76061 strain and the strains obtained from said strains by selection or mutation when a high glucoamylase activity is desired. The ATCC 76061 strain is particularly preferred.
DETAILED DESCRIPTION
When a high glucoamylase activity is desired, the wheat bran used as starting material should be a non-starch-free bran. Apart from this restriction, any bran may be used. However, the bran preferably comprises a significant proportion (at least 40 wt. %) of particles of less than 1 mm.
The characteristics of two suitable brans are given below by way of illustration.
Bran A
Bran B
Characteristics
Moisture (%)
12.3
19.5
Protein content (% WM*)
13.8
14.8
Starch content (% WM*)
24.6
21.3
Particle size
>1.25 mm
53.9
0.7
from 1.0 to 1.25 mm
8.1
1.3
from 0.5 to 1.0 mm
33.3
68.2
from 0.25 to 0.5 mm
3.7
24.6
from 0.16 to 0.25 mm
0.3
2.6
<0.16 mm
0.7
2.6
% WM = % relative to the wet matter.
The wheat bran should be moistened and heat-treated in order to pasteurize it or to sterilize it. It is advantageous that the heat treatment does not precede the moistening because poor fermentation results have been obtained if the bran is heat-treated before moistening. The heat treatment may consist in heating, for example, in an autoclave. An autoclave treatment of 20 min at 120~121° C. has proved highly satisfactory, but less severe conditions (pasteurization at 105° C. for 15 min in an oven) are also suitable. It is also possible to carry out the heat treatment of the bran by injecting steam into it, which may make it possible to moisten the bran simultaneously.
The pH may advantageously be adjusted during moistening in the range from 4 to 5.5 in order to improve the pasteurizing effect of the heat treatment and the initiation of the desired fermentation.
In addition to its sterilizing function, the effect of the heat treatment is to promote gelatinization of the starch contained in the wheat bran and therefore the availability of this substrate for the fungus
Aspergillus niger,
which allows more effective fermentation.
The moistening of the bran is important because the water content influences the performance of the fermentation. The initial water content of the bran is initially adjusted to 50-60%, preferably 50-55%, of the total mass of the bran and of the water and it is substantially maintained in this range during fermentation, for example by periodically supplying water in order to compensate for the loss of water from the medium. The expression “substantially maintained” means that it is acceptable for the moisture level to take a value which varies slightly (±5% units) from the range 50-60% during a relatively brief period between two successive adjustments of the moisture level or at the end of fermentation. It is advantageous, in any case, not to drop below a moisture level of 45%. The moisture level of the culture medium tends to decrease during the culture through evaporation due to the increase in temperature generated by the fungal growth, said medium being a poor heat conductor. The quality of the water used also plays a significant role. Good quality running water or distilled water may be used.
The inoculation of the wheat bran may be performed with any appropriate inoculum. Persons skilled in the art know many ways of preparing a suitable inoculum from

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