FR2504150A1 - GLUCOSE PRODUCTION - Google Patents

Abstract

IMPROVED PROCESS FOR ENZYMATIC PRODUCTION OF GLUCOSE FROM A LIQUEFIED STARCH SOLUTION. IT CONSISTS OF SUBMITTING A SOLUTION OF LIQUEFIED STARCH, WITH ED LESS THAN 15, TO THE COMBINED ACTION OF A GLUCOAMYLASE AND AN ISOAMYLASE, WITH A PH INTERVAL BETWEEN 3.5 AND 4.8, DURING 30 A 50 HOURS, AT A TEMPERATURE OF 50 TO 60C. THUS EASILY OBTAINED, WITH HIGH YIELDS, A VERY PURE GLUCOSE.

Description

The present invention relates to an improved process

  for the enzymatic production of glucose, from

a solution of liquefied starch.

  In a classical way, sacchari-

  glucose starch, as follows: a suspension

  starch is liquefied with an acid or a <-amy-

  lase, then undergoes the action of a glucoamy-

  to give a saccharified starch solution with a glucose content of up to 90 to 94%,

  dry solid (all percentages referred to below are

  tend to dry solid) This solution is decolorized with activated carbon, deionized by means of an ion exchanger, concentrated, and crystallized; and the resulting mass is then in high glucose powder, or separated into crystallized glucose and mother liquor, according to subsequent use. Recently, in order to enhance the glucose content

  in the saccharified starch solution, several improvements

  The Japanese patent, published under No. 29570/79, suggests that during the course of saccharification

  from starch to glucose, thanks to a combined enzymatic system

  taking glucoamylase and amylo-1,6-glucosidase, a solution of liquefied starch, at ED 15 to 20, is subjected to the action

  of the enzymatic system in a range of p H inclusive

  5 and 6.7, an interval in which suffi-

  the reversible reaction of glucoamylase.

  pones, published under No. 23 894/81, indicates that, during the saccharification of a liquefied starch into glucose by means of glucoamylase, it is desirable to use, in combination with glucoamylase, a "-1.6- Bacillus glucosidase,

  in an interval of p H between 5 and 7

  Poles, published under No. 23 895/81, also proposes, when

  saccharification of a liquefied starch with a glucose

  amylase, to let a -1,6-glucosidase act in a

  p-val of 5 to 7, on a partially saccharified starch solution, in which the degree of saccharification

amounts to 30 to 80%.

  However, although in all these suggestions the saccharifications were carried out at a higher level.

  at 5, we find that the solutions of saccharified starch re-

  sultants, high in glucose, prepared on the

  industrialists, according to one of these suggestions, are

  life expectancy of a brownish reaction

  and microbial contamination, under the conditions

  saccharification, that is to say temperatures

  500 to 60 ° C and duration of 40 to 90 hours.

  The present invention avoids these inconveniences.

  from the prior art, and to obtain a solution of

  saccharified midon with high glucose content, thanks to

  use of a p H less than 5 during the operation of

saccharification.

  The novel process according to the invention consists of

  to convert a suspension of starch into a starch solution

  queued at low ED, then to subject this solution to the action of a glucoamylase (EC 3,2,1,3) and an isoamylase (EC 3,2,

  1.68), in a range of p H between 3.5 and 4.8.

  The starch used according to the invention can come from roots or tubers of potato, sweet potato and cassava, as well as grains of maize, rice or wheat. Other sources of starch can also be used.

  gruel or tuber tips with a high amicable

Don.

  As regards the concentration of suspen-

  starch is suitable for any concentration, provided that the

  midon is sufficiently liquefied and dispersed; concentrations of 10 to 50% are usually used, preferably

between 20 and 40%.

  The liquefaction of the starch suspension can

  be carried out according to a conventional method, and the solution

  The resultant liquefied starch is then subjected to the enzymatic action of a glucoamylase and an isoamylase, at a temperature of the order of 50 to 60 ° C., and to an inclusive pH.

  between 3.5 and 4.8 During the saccharifica-

  In the case of the liquefied starch solution, the isoamylase must be added before the ED of this solution reaches a maximum of 15, preferably 10.

  Glucoamylases (EC 3,2,1,3) that can be used to

  according to the invention, are glucoamylases which act

  on the liquefied starch solution in a pH range of 3.5 to 4.7, and which release glucose units; in general, because of their lower optimum pH (4 to 4.5), commercial glucoamylases from molds, such as

  Rhizopus and more particularly of the genus Aspergillus

read.

  In accordance with the invention, the

  amylases (EC 3,2,1,68) from bacteria, such as

  those of the genus Flavobacterium, Cytophaga and especially Pseu-

domonas.

  When the saccharification is over, the

  The resulting solution is heated to inactivate the enzymes

  present, and then purified, for example by filtering

  discoloration with activated charcoal, and / or deionizing

  ion exchange; Finally, it is concentrated to give a syrup of amido 4 with a high glucose content. The glucose content of this syrup is very high, of the order of

-98%.

  Unlike saccharification according to

  conventional processes, which is performed at a p H equal or higher than

  5, the saccharification according to the invention, which is

  in a range of pH between 3.5 and 4.8 by means of a combination of glucoamylase and isoamylase, has the following characteristics: shortening

  the duration of saccharification, reduction of contamina-

  microbial growth and browning reaction during

  saccharification and, consequently, purifica-

  The solution of saccharified starch is subsequently made much easier. It follows that it is easy to produce on an industrial scale the production of

  saccharified starch solution with high glucose content.

  It follows from the advantages described above that the present invention can be advantageously used for the production of crystallized glucose, fructose by isomerization of glucose, sorbitol by hydrogenation of glucose, and other products such as ethanol and monosodium L-glutamate, which are obtained by fermentation of glucose,

  in order to further increase the purity and / or the yield.

  In this description, the enzyme activities

are tried as follows.

  1) Glucoamylase (EC 3,2,1,3); the unit of glucoamylase activity is defined as the amount of the enzyme that

  releases 1 mg of glucose in 3 minutes at 40 C in a mixture of

  reaction comprising 5 ml of aqueous solution of amine

  soluble donation at 1% w / w, 4 ml of acetic buffer solution

  1 M (p H 4.5), and 1 ml of a glucoamylase solution.

  2) Pullulanase (EC 3,2,1,41) or isoamylase (EC 3,2,1,68); the unit of pullulanase or isoamylase is defined as

  the amount of the enzyme that increases the absorptive

  At a rate of 610 nm of 0.01 per hour. More specifically, to the solution comprising 5 ml of 1% w / w aqueous water soluble waxy rice starch solution, 1 ml of 0.5M acetate buffer solution is added. whose p H is adjusted to 6

  for pullulanase, or 3.5 for isoamylase, and this

  The mixture is incubated at 40.degree. C. for 1 hour. 1 ml of this reaction mixture is then taken off, 1 ml of 0.01 M aqueous I-KI solution is added thereto, and subsequently of 504 H 2 0.02 N, to obtain a final volume of 25 ml 15 minutes after the addition of the sulfuric acid, the absorbance of the mixture is measured at a wavelength of 610 nm,

with a cell of 1 cm.

  The glucose content in the starch solution

  saccharified is determined by classical analysis by

  matography on paper, and it is expressed in percent

  weight, relative to the total amount of sugars.

  Several embodiments, not limited to

  of the invention are described in the examples below.

EXAMPLE 1

  To a starch slurry of 28% w / w is added calcium carbonate at a rate of 0.05% based on dry solid starch, as well as a commercially available "Bacillus Licheniformis" -amylase "Termamyl. 6 OL "(Novo Industri

  A / S, Copenhagen) at a rate of 0.15% relative to the starch

  dry, and this mixture is liquefied at 98 ° C. for 30 minutes.

  Then, it is brought back to ambient conditions, which gives a liquefied starch solution with an ED = 4. After having rapidly adjusted the pH to 4.5 and cooled to 55 ° C., 1 liter flasks are placed in water. withdrawals of 500

  ml of this solution, to which 4 units of glu

  Aspergillus coamylase "XL-128" (Nagase & Company, LTD,

  Osaka), and 150 units of Enterobacter pullulanase or

  Pseudomonas soamylase (Hayashibara Biochemical Labora-

  tories Inc, Japan) These samples are then submitted

  at 40 hours of saccharification at pH 4.5 and 55 C.

  The control experiment is conducted simultaneously, and the sac-

  charification is carried out identically

  that neither pullulanase nor isoamylase is involved.

  When saccharification is complete, the glucose levels of the starch solution samples are determined.

  saccharified The results are given in Table 1.

TABLE 1

  Sucrose Enzyme Tested Glucose Content (%) Glucoamylase 93.9 Control Glucoamylase + Pullulanase 94.0 Control Glucoamylase + Isoamylase 97.4 Invention

  The experimental results, reported in this table, con-

  that it is easy to obtain an amicable solution

  saccharified gift with a very high glucose content, by saccha-

  purification of a liquefied starch solution by the combined action of glucoamylase and isoamylase at pH 4.5.

EXAMPLE 2

  Samples of 500 ml of a solution of

  ED = 4, obtained as in Example 1, are placed in flasks of 1 liter, and quickly adjusted their p H respectively 3, 3.5, 3.8, 4, 4.5, 4.8 , 5.1, 5.5 and 6 with hydrochloric acid, and is cooled to

   C Then we add to these samples, as in the example

  1, glucoamylase and isoamylase, and incubated at 50 C, at the specified pH, for 48 hours, in order to

  perform enzymatic saccharification.

  At the same time, the control experiment is carried out

  re identical, but in the absence of isoamylase.

  When saccharification is complete, eqglucose contents are determined, as well as the degree of staining and

  microbial contamination, in samples of

  The results are shown in the table below.

table 2.

  As this table shows, the experimental results confirm that saccharification carried out by means of a

  combination of glucoamylase and isoamylase, in a

  p H between 3.5 and 4.8, results in

  saccharified starch / with the highest glucose levels,

  and that, at p H equal to or greater than 5.1, the solutions of

  saccharified gift are very sensitive to the reactivity

  and microbial contamination.

  (See Table 2 on the next page).

TABLE 2

  Enzyme p H Glucose Content Relative Degree of Degree of Contamination (%) Microbial Staining

3.0 92.1 94 (-) *

Glucoamylase 3.5 95.5 96 (-) **

3.8 96.4 96 (-) **

4.0 97.6 98 (-) **

+ 4.5 97.2 100 (-) **

4.8 96.0 103 (-) **

, 1 93.8 118 (*

Isoamylase 5.5 92.0 135 (+)

6.0 86.2 254 (+) *

  _________________________________________________________________________________________

  Glucoamylase 4.5 93.9 98 (- * or LM (Table 2 continued) Note:

  1) "The relative degree of coloring" is calculated by the

  quation: relative degree of staining (%) = B x 100 A where A is the absorbance of the filtrate, at the wavelength of 420 nm, with a 1 cm cell, which is obtained by saccharification of a solution of liquefied starch

  p H 4,5 with a combination of glucoamylase and iso-

  amylase, followed by filtration of the resulting solution of saccharified starch by means of filter paper; and B is the absorption capacity obtained as for A, with the exception of

  the solution of liquefied starch is saccharified to another

  be leveled p H, before being set to p H 4,5.

  2) "The degree of microbial contamination" is deter-

  mined after completion of saccharification, by the measure

  number of microbial colonies in the solution of

  saccharified gift; (+) indicates that more than 1000 colonies are found in 1 ml of saccharified starch solution, (+) between 100 and 1000 colonies, and () less than 100 colonies

in 1 ml.

  3) () * denotes a control product and () ** a product

according to the invention.

EXAMPLE 3

  Different ED samples, 35% w / w liquefied tapioca starch solution, obtained as in

  Example 1 are adjusted to pH 4.5 with hydrochloric acid.

  drique and cooled rapidly to 60 ° C. Then 5 units of a commercial Aspergillus glucoamylase "Amyloglucosidase 150 L" (Novo Industri A / S) are added to these samples.

  Denmark), and 75 units of Pseudomonas isoamylase (Haya-

  Shibara Biochemical Laboratories Inc., Japan), and

  incubate these mixtures at 60 ° C and pH 4.5 for 36 hours,

  end of achieving enzymatic saccharification.

  At the same time, the control experiment is carried out

  same, but in the absence of isoamylase.

  When saccharification is complete, the glucose contents in the resulting saccharified starch solution samples are determined. The results are reported.

Table 3.

  As this table shows, the experimental results

  confirm that when starch is converted to glu-

  using a combination of glucoamylase and iso-

  amylase, to obtain a solution of saccharified starch with the highest glucose content, it is necessary to add

  the starting liquefied starch solution, at less than 15.

TABLE 3

  E.D Glucoamylase Glucoamylase _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ + Isoamylase

1.9 92.5% * 96.2% **

4.2 93.8% * 97.0% **

8.8 93.7% * 96.7% **

, 0 93.3% * 96.2% **

14.2 92.8% * 95.1% **

19.6 92.4% * 93.8% *

21.4 92.1% * 93.2% *

  Note: *), witness; **), the present invention.

  Note: The conventional abbreviation, ED, used in the

  preceding section, means the equivalent of dextrin;

  enzymes their Enzyme Classification Number.

  my, EC is given in parentheses.

Claims (4)

claims   1 Process for producing glucose, which consists of   saccharify liquefied starch with a combination of   glucoamylase and a disintegrating enzyme,   in that a solution of amidorediol is   ED less than 15, with the conjugated action of a glucoamylase and an isoamylase, at a pH between 3.5 and 4.8. 2 Process according to claim 1, characterized in that the isoamylase comes from the genera Flavobacterium, Cytophaga or Pseudomonas.   3. Process according to one of claims 1 or 2, characterized   in that the glucoamylase originates from the genera Rhizo- pus or Aspergillus.   4. Process according to one of claims 1 to 3,   in that the liquefied starch solution has an equal ED or less than 10.   Method according to one of claims 1 to 4,   in that the liquefied starch solution is subjected   the enzymatic actions of glucoamylase and isoamy-   lase at a temperature between 50 and 60 C, during at 50 hours.