JPS5831991A - Immobilization of mold with alginate - Google Patents

Abstract

PURPOSE:To obtain an immobilized mold-containing substance having high activity and mechanical strength, by treating an alginate-containing solution suspending the mold with an Al salt-containing crosslinking solution under specific conditions. CONSTITUTION:A mold, for example, about 24g wet and swollon wine yeast with a water content of 75wt% is suspended in about 36g of about 1.5wt% aqueous solution of sodium alginate, potassium alginate or ammonium alginate. The suspension is added dropwise through a nozzle to about 0.05gmol/l aqueous solution of Al salt such as Al2(SO4)2, KAl(SO4)2, AlCl3, Al (NO3)3, etc. as a crosslink ing solution in a PH of 3.1-4.5, to give a mold-containing particle gel immobilized with the alginate. In order to make the gel particle hard, about 20mol of the crosslinking solution is added to it with controlling the pH to 2.4-3. After the gel is allowed to stand for 24hr at 5 deg.C, about 22g gel particle having a particle diameter of about 1mm. is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for immobilizing bacterial cells; or Aluminum J, in which bacterial cells are suspended in an ammonium salt aqueous solution;
This invention relates to a method of treating with salt-containing cross-linked NK and immobilizing bacterial cells with aluminum alginate.

Regarding the conventional technique for immobilizing bacterial cells, the recently developed encapsulated bacterial cells immobilized with the potassium salt of carrageenan are susceptible to mechanical shock and are difficult to escape from the encapsulated bacterial cells. There are many 41 leaks. Therefore, it is no exaggeration to say that the alcohol fermentation method using this immobilized bacterial enclosing body is vastly different from the conventional method using live yeast. It is necessary to carry out the test in an environment near or near the ground.

In addition, the present inventor's patent application No. 11 Sho 55-141820 discloses that the bacterial cells are prepared by a two-step method, that is, first, they are immobilized with calcium alginate. This was obtained by creating an inclusion complex, and then replacing some of the calcium ions with V minilum ions through ion exchange.When an alginate solution in which bacterial cells were suspended was directly treated with an aluminum salt, pT-
The activity of the bacterial cells decreases due to the decrease in I, and on the other hand, if the concentration of aluminum salt is reduced to reduce the pH drop, the activity of the bacterial cells remains, but the mechanical strength becomes insufficient. Is it possible to solve the shortcomings in this way? 1) Alginate aluminum J. Calcillano,
If the solid ′, iy seedling body i1”i f, I< is held once a month,
I) Alcohol fermentation occurs actively even under 112.8~30, which is an unfavorable environment if fermentation 'l: 1+i' is 1'1]) 11 , ('1°J Application 5G-43639). The U method for creating such an effective fixed '+-' i law field is 11
, the use of the element 14 of depletion of ζ 51 is less ii1. It is desirable that the middle of the 11th stage is in i'+i'i. In Toro, the 11 substances of the 11 salt complexes are so strong that they are so troublesome.
``1 (so, how can I do it in a single step''): In Rinuma I, there is a 1.
(Human being comprehensive 1 I short 11.1: Between, and 1”
Is it possible to produce it on an industrial scale?

In other words, if you use the present invention, you can use the bacteria f4.%7'iii al, V salt-soluble Yfk, not aluminum, but even the prl containing aluminum. 1-4
．． 5, f'I-1, and then pH 2. 4-3
．． It was discovered that if γ of 0: ('lI +!b1A), an active and mechanically strong immobilized bacterial cell enclosing body could be obtained.

When carrying out the present invention, the concentration of the aluminum salt is, for example, A. ]-2(SO4)g is 0
01qrnr)-1
-1 gelatinization size after being released into a tube) 1. How can immobilized enzyme fJ be obtained without dividing pT1 conditions into three stages? 1 (C, but the bacterial cells tend to be inactivated, and it takes a long time for RJC.
1 Mechanical strength is also weak. Such a method is too simple to produce 71 immobilized bacterial cells for alcohol production on an industrial scale.

A. In closing, 1, - cross-linking liquid mouth] As aluminum salts, VJ+fe acid aluminum, sulfate, aluminum, ammonium, nitrate, aluminum, aluminum chloride, lactic acid, aluminum, and aluminum salts. Any water-soluble inorganic or organic compounds such as aluminum chloride, aluminum ammonium lift acid, Mi:
Aluminum/potassium acid is an easy f song II''/: l and Ibe Sui. r: I'i-( , 1,
1,! , becomes 3,/r. For example, 1(Δl (:;(1・
I) 2 solution (fn is 0.05 [110 rezl l+
:'a t(y has J? and pI+8.2.

Inside this is 1 J Mrs. 1 Nirvin Engineering 1 Te 1 F 4
\! Four plays,) shi.

Alginic acid 1-riuno rfk'? t: In the life of a person, 14C is 7■:.

Susutosu1nt and Alumiuno's i''-Nben-1
1i! ! The reaction progresses and gels, and the aluminum in the middle of the night:-UJ
．． ．． , (-4 n concentration is low 1 · eyelids, 1 in rl) [1 is 1 + f1.

Alginate, 1' life, 1' white, 11: (I
RI turbidity - J-ru I〈Δl (SOI) 2 melting (currently I put it in the cutting, the same ()t na・11 is 1,・koru.

8 (Cell','06Na),, l rl(△
l (,':(')-+)2→(' ((:6 11
706)3 Δ])n I-stir l<21:()1 I
(-F1 111.2::04 Immobilized bacterial enclosing body near the surface of the gel (G/I/fI) progresses, and the inside of the gel is also ion-exchanged. If the vicinity of the surface turns into a 1- state, even if 11 of the liquid body is reduced to 28, 1) does not change from 11 to 32.
Tsuda inclusive +W (7 compared to 4-)1/ :I-
/l/ /l 11?1'l+ is unchanged, Menki f1・
pH'fi; 2. 5 V]-Tena J, produces alcohol and proliferates within immobilized inclusion bodies. According to the present invention: Bacteria f immobilized in the immobilization package prepared by
4,-11 is approximately 200 to 360 Q (dry weight) per inclusion body 16, and when solidified with carrageenan or polyacrylamide, the bacterial cell retention is much greater. It has a high physical strength, high alcohol fermentation productivity, and low [,,l■r
However, it has the ability to proliferate.

Hard 5;! The amount of bacterial cells to be converted is 2% of the solution of sodium alginate, kaliuno, aluminium, or a mixture of these.
The ratio of 1J l'2 and 1[1 to the bacterial cells suspended therein can be reduced by /1↓.

For example, if we take the genus Mycenu as an example,
When using 1009, 1.5% (by weight) of alginic acid triuno water-soluble i (1 150 g), approximately 100 ml of aluminum alginate immobilized inclusion body is 4
71, and the immobilized bacterial cells p1- (dry weight) was 250q per 11 immobilized inclusion bodies.

Bacteria f
・, ammonia J・, and their mixtures can be used at any concentration (
'U, it's fine, but 1 to 1.5 'y'o (middle 111) water In (<'e force; iy Jil easy.

When carrying out alcohol f1.1 fermentation, (J,: physical 1, J of immobilized aluminum 14)
:bi f[; cheap 1 go 1' of scientific properties, ke supply tl! i
tj is achieved by adding aluminum ion to Y+'Q. Prefecture l (of which Wenzhou mandarin oranges, -nu super, if left unattended, 15n・substances will settle in the next "rs": '? + [
ll, close the pipe: l, 1
For example, if you add 1 to 2 q/l of aluminum sulfate (17) to a juice containing 1.5 M of sucrose (17), it will quickly settle and solve the problem of blocked I/nozzles. The excess aluminum sulfate (1') serves to stabilize the aluminum alginate immobilized inclusion for a period of 1.2 years.

There are no particular restrictions on the fungi to be used, including all members of the genus Tofucharomyces, such as Eharitsuka + I Mitonu Carlsbergensis, Schizoritsuka 11 and all members of the genus Schizoritsuka [I Mishi]. Nis bombe, all members of the genus Candeida, e.g., all members of the genus Candeida, e.g. all, e.g. Gluconobacter zapuogicidans, all members of the genus Acetobacter, e.g. Acetobacter
Aceti, all of the genus Shootmonas, e.g.
-)"Sled-y, obalis, etc. can be used.

Examples and reference examples are given in Section -F to explain the ili'j method of aluminum alginate-immobilized yeast cell inclusion particle gel and the alcohol fermentation method using the same.

Example 1 Using bacterial cells 1 and twinic acid r1 (Zatsukalomycenu cervicii 0C2-2), culture was carried out by shaking in Ylφ soil, and the bacteria was centrifuged to obtain a 75% water content (by weight). i71: Wet ffi' containing ) fermentation f'9
24 g (24 g) was suspended in 1.5 (weight) 6 μl of an aqueous solution of 1-lium alginate ff86ti. This 1 suspension is passed through a nozzle, and the crosslinking liquid o, o 1g+10]-0
71 (= 2.6 q/l) KA 1- (S 04
) 2 solution 1 ((800ml (pTI8.6)) When dropped into 800 ml (pTI8.6), alginate aluminum immobilized t1\1', which is ionized only in the vicinity of the surface, forms a gel containing engineered bacterial cells. Next, 0.1 gmal−0 was added to the solution containing this 1′ gel while controlling the pL+3.14.
/l KAl,, (SO4)21 B, 6 m,
1 g+11010/l of KAl,
(304) 215 m, l was added dropwise while controlling the pH to 2, 6, and 5°C, and the mixture was incubated at 5°C for 1 hour in the M apparatus. As a result f
Y2RR solid, i2 fermentation 1'', 1 bacterial cell inclusion particle gel 22ti is T-5). .

Reference Example 1 Add Δ12 (F18) to pTTL2 mandarin juice
S04)! Add 1 to make pT'+ 2.8 and add bukomandarin juice (medium 11.1i toshite),, 6 gmal
, 0/l) 800 m, t and 0.5 q of non-enclosed particles washed with physiological saline No. 1 (1 hour) were placed in a square flask and alcoholic fermentation was carried out at 30°0 with shaking.

84q/l after 24 hr, 72tl work = 12oq/l after
7) v Co/l'a degrees. Towing 17 to the outside of the immobilized body 1'! (The number is 7.8 x 1 for i'j1 person)
05/m, t, after is 1.9 X 1 o'/m,
It became 1. In pT13.2, 78 q after 24 hr'
/l, 72 l'+r then 12 (1/l of 1)
-le = t+:Is IB:', and the number of leaking bacteria is 1.9 x 105/nbl, 2. OX
It was 108/ml. That is, the results of the method of the present invention conducted at (■)■12.8 are significantly different in both alcohol productivity and bacterial cell productivity when compared to those conducted at pV■3.2. Moreover, 90% of leaked bacterial cells at pH 2.8
%L=J, -1- was a viable bacterial cell.

Example 2 i7.1 cells were cultured using wine acid/"J (Zatsucharomyces cerevisiae QC-2) with shaking in a 1φ medium, and 75% of the moisture (FT
lrl-i: ) Wet fermentation containing f'124q'i1.
Suspended in 5% (weight) sodium alginate aqueous solution i & 86q, this suspension was passed through a nozzle, and the crosslinking liquid 0
, OIQmO]-, c]/l (-= 8.42 q/
l) Aqueous solution of 2 (SO4) 3 (pT (pT). Next, to harden the gel particles, the A IQ (S 0
4) Add 320 ml and leave it at 5°C overnight.

I'Blu+ game) Vre "f reservation 229 got.

Reference example 2 ``2 il! 11111 gel age)
”l completed 05 Rik 11. J Ko food Ibu, +1
After soaking with water for 51 degrees, it is about 189 A.
12 (',,'',,(1,+)Add 3 and get pT'
Put the mandarin orange juice (medium IJli) into f 2.8 (1.ri & rll (') -, 1, (4/l) and add IB (tocho alcohol intoxication fermentation (-1). 3
0°C) After 2411r Alcohol J Negative 30ti/1 mountain 11 bacteria count 9X] 05 After 7211r -f / L / Co / l / 713 degrees 1
20q/1IA1" + number of surrogate bacteria 3.lX108 one) H8, 2 is 72br after 7) = 1-) then 1 A JQr: 1 2 0 q/l number of bacteria 1
．． 'I X 108, i.e. T) IT2.8
and 3.2, there is no big difference in productivity.

Alcohol production and proliferation also occur in pI-I2.5, as shown below.

After 50hrs Alcohol consumption: 52t
i/l leakage bacteria count 8X107 Example 3 (11) Different from the example described in 11), a disk-shaped Zatsuka "I Mrs. Servicier W" with a frame of 100 mesh stainless steel wire mesh and a diameter of 9 cm and a thickness of 3 cm was used. -3WE, an example in which Bacillus cells were comprehensively immobilized and alcohol fermentation was performed.

Fermentation 1iJ bacterial cells were cultured in a tank on Ylφ"7"τ soil, and 10 g of wet fl'F f No. (moisture 80%) was collected at 1.5
% (weight @) of sodium alginate, diameter 9.5 cm
Place in a 2g molding container with a depth of 5mm. Add a frame made of 100 mesh wire mesh to this. Next, while cooling the outside of the molded container with ice water, 200 m1 of 0.059 mo]-e
/lK A E-(,',E O4)! Gently pour 2 armpits, turn over for 2 hours and leave for another 2 hours. This disk-shaped aluminum alginate-immobilized yeast cell enclosing body was taken out from the liquid, and 0.0% was applied to its surface. I QTnOJ-e/l(D
Spray KAI (SO4)2 aqueous solution. Furthermore, 0.59 moE-e/l', Al (SO4)
2 aqueous solution f, (y−y”v−t le.

Reference example 3 Take the 15 disks obtained in this way on a horizontal axis (12). Make sure that the soil is not immersed in the liquid, and add molasses (1.5
Add sulfuric acid and 1S/Z aluminum sulfate to M) and add pT'
Supply f2゜8 liquid to 6, disk rotation speed 1100 rpm
, 30°C, 1V + 2 gas is applied to the soil part of the bioreactor, and the feed liquid is supplied to the lower part.It can be operated continuously for more than 1 month without any trouble. is 10
It contained 0-110q/l of alcohol.

Example 4 Alcoholic acid fJ 12 Q cultured by shaking in molasses medium
(Dry i13i (semi-) is suspended in the armpit 11, under stirring 171
Then gradually add KAI (SO4) 2 . This pT
160q of 1.5% (by weight) ammonium alginate is added dropwise to the suspension of 8.2 through a nozzle.

To further harden the purified gel particles T) TI2.
Igmo]-e/1(-=841/l
) (add more D ammonium sulfate, o, 5t, 1
Leave at 5°C overnight. The resulting aluminum alginate entrapping immobilization enzyme fJr) with a diameter of about 211R was taken out and thoroughly washed with cold physiological saline containing 1.59 mO'le/l of aluminum sulfate to immobilize it. 90g of yeast particles is 11).The concentration of bacteria in the particles is 2
It was 0-100g (dry-17I)/7.

Reference Example 4 90 g of these particles were packed into a 3-stage, two-bottom conical bioreactor with an internal volume of 480 m and 1, and the pH was adjusted to 2 with a N2 guff of 0.08 V/V/m and sulfuric acid from the bottom of the bioreactor.
48 m of molasses with a sugar content of 18%, l/A
Enter that. The reaction is carried out at 30°C, and gas and liquid are continuously pumped out of the bioreactor.

In this continuous alcohol fermentation operation, during steady operation, the delivery liquid contains 7.2-8.0 (w/v)% alcohol.

Applicant: Hin Shuzo Co., Ltd.

Claims (1)

[Claims] If the bacteria are suspended in an aqueous solution of alginic acid (alginic acid), potassium or ammonium salt (alginic acid), potassium or ammonium salt, or d-alginic acid (alginic acid), potassium or ammonium J, or an aqueous salt solution (lr). 1) H3, 1-4.
A method of immobilizing bacteria f4. by treating under the conditions of No. 5 and then under the conditions of p T72.4 to 3.0.