JPH0576362A - Production of lysozyme-containing particle - Google Patents

Abstract

(57) [Summary] [Purpose] Although lysozyme is an extremely useful antibacterial agent,
The use of dried lysozyme is limited by the propensity to dust and the difficulty of dissolving it in water. Provided is a method for making dry, non-dusting, lysozyme-containing particles and a dry, non-dusting particle comprising a homogeneous mixture of lysozyme and other active and inactive ingredients. An aqueous slurry of lysozyme is sprayed with core particles into a reaction chamber of a fluidized bed reactor, thereby evaporating residual water to leave dry lysozyme coated on the particulate core material, thereby lysozyme. A manufacturing method is disclosed which comprises providing particles containing a.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a dry, dust-free lysozyme-containing particle and a dry, dust-free particle containing a homogeneous mixture of lysozyme and other active and inactive ingredients. A method for producing particles. The particles are processed or compounded food products such as cheese, communated meat products, salads and soups; antibacterial dips, sprays and other therapeutic ingredients, and various other food products. , Especially useful in cosmetics, pharmaceuticals and industrial applications.

[0002]

BACKGROUND OF THE INVENTION Lysozyme, which is now officially described as N-acetylhexosaminidase, is a product of the enzyme committee (Co
mmission on Enzyme) gave the designation of 3.2.1.17. This enzyme with a molecular weight of about 14,300 to 14,600 and an isoelectric point at pH 10.7 can be obtained by crystallization from egg albumin.

Lysozyme is a long, complex sugar molecule found in the cell walls of many live bacterial cells, β (1) between N-acetylmuramic acid and N-acetylglucosamine.
-4) Named for its ability to degrade or lyse the cell wall of bacterial cells by virtue of the ability to cleave or hydrolyze bonds. Due to the nature of the cell wall, Gram-positive bacteria are most sensitive to lysozyme. Because of its antibacterial activity, lysozyme has been described as useful growth Clostridium food product (Clostridia) for suppressing food preservative. Carini
i) et al., Microbiologie-Aliments-Nutrition, 198.
5, pp. 299-320.

[0004]

Although lysozyme is a very useful antibacterial agent, the use of dried lysozyme was somewhat limited by its propensity for dusting and its difficulty in dissolving in water. These are serious drawbacks because of the allergenic nature of proteins that can occur when inhaled into the lungs. Chronic exposure to such dust can result in serious immune reactions. Furthermore, the currently available dry lysozyme is difficult to redissolve in water, extending the time required to work with this material.

Food Science and Nutrition, 26, 4
Edition (1988), pages 359-395, CRC Critical Re
-In views, Procter and Cunningham report that lysozyme is thermally stable in acidic solution and withstands 100 ° C with little loss of activity. However, the proctor, etc. can be used within 30 minutes in a borate buffer solution of pH 7.9 within 70 minutes.
It is argued that 25% of lysozyme is inactivated at 0 ° C.
This paper also states that lysozyme in egg white decomposes at 65 ° C at pH 8.5, but at pH 5 and 60 ° C
No loss of activity occurred within 60 minutes. Also, lysozyme has a pH of 6 at 62.5 ° C rather than in egg white.
It was reported that it was found to be more than 50 times more heat stable in the phosphate buffer of 5.

The activity of lysozyme is also affected during its processing. Procter et al., Pages 365-366, report that the type of container used to store lysozyme can affect its activity. That is, in an experiment on the effect of Pyrex (registered trademark), polypropylene and polyethylene containers, refrigeration in Pyrex (registered trademark) and polypropylene containers 24
It was found that over time, the concentration of lysozyme was reduced by 40%. In other studies, lysozyme was found to be poorly reproducible in its activity due to its attachment to glass containers. Good et al., US Pat. No. 4,684
No. 9,297, a granular core material was introduced into the manufacturing chamber of a fluidized bed reactor in which an aqueous slurry of enzyme was sprayed onto the particles, and a dry layer of enzyme was formed on the core particles upon evaporation of water. The method for producing enzyme particles without dusting is described. Goode et al. Describe an air inlet temperature of at least 50 ° C. for treating a thermally stable enzyme, but nothing about how thermally and environmentally unstable lysozyme is treated in a similar manner. No guarantee is given.

[0007]

SUMMARY OF THE INVENTION The present invention is coated on a particulate core material by spraying an aqueous slurry of lysozyme along with core particles into a manufacturing chamber of a fluidized bed reactor to evaporate residual water. It relates to a process for producing dust-free lysozyme-containing particles, which comprises leaving dry lysozyme, thereby providing particles containing lysozyme.

[0008]

DETAILED DESCRIPTION OF THE INVENTION The lysozyme-containing particles of the present invention are formed in the reaction chamber of a fluidized bed reactor. Typically such devices have a circular grid with a porous grid at the bottom, with the top opening to open like an umbrella towards a conical expansion chamber with a larger diameter than the circular product chamber. It is composed of a dryer including a storage room. In operation, as the velocity of the air rising through the chamber increases, the "fluidization" and "fluidization" is reached as the particles on the porous lattice reach a point where they are suspended in a stream of air like a fluid. It is called a floor dryer. The particles are lifted from the product chamber by the rising force of the air entering the expansion chamber, where the air expands and the rising force per unit area is reduced. As a result, the particles fall back into the product chamber and the circulation work is repeated.

The initial stage of the process of the present invention is the introduction of an aqueous slurry of lysozyme into the reaction chamber of a fluidized bed reactor,
And suspending it in an air stream. A large amount of core particles is injected with the lysozyme slurry. Lysozyme is repeatedly contacted with foreign matter at elevated temperatures during particle formation in a fluidized bed reactor. Generally, the air temperature inside the reactor is 40 ° C to 60 ° C.

The core particles are highly hydratable substances, ie
It consists of a substance that is easily dispersible or soluble in water.
The core material must be solubilized by dispersing (disintegrating and losing its cohesiveness) or by transferring to a true solution. Clay (bentonite, kaolin), non-pareil and agglomerated potato starch are considered dispersible. Nonpareils are spherical particles consisting of a solid core that is spherically rounded by a layer of powder bonded to the core in a rotating spherical container. Salt particles (NaCl crystals, NaCl rock salt, NaHC
O ₃ ) is soluble. Also suitable are agglomerated trisodium citrate, NaCl flake crystallized in a crystallization dish, bentonite granules and granules, bentonite / kaolin / diatomaceous earth pelletized granules, and sodium citrate crystals. The core particles consist of substances that do not dissolve during the subsequent spraying process, and are generally particles whose longest dimension is 150 to 2,000 μm (100 mesh to 10 mesh on US standard sieve system). is there.

Generally, lysozyme or lysozyme and other active or inactive substances will be 10 to 25% solids (w / w).
w) dispersed in water to a concentration. Any optional binder,
Dispersions containing salts or plasticizers are pumped and have a low enough viscosity (typically 10 to 5,000 cp at room temperature) to be atomized as an effective spray coating solution.
s). The lysozyme preparation is applied to the surface of the core by fluidizing the core particles in a stream of air, whereby a solution containing lysozyme and optionally other solids is atomized and sprayed into a fluidized bed. The atomized droplets come into contact with the surface of the core particle, and when the water evaporates, a solid thin film is attached to the surface of the particle.

There are numerous reports in the literature describing useful combinations of lysozyme and other active ingredients. The present invention can be used to produce a dry, non-dusting, homogeneous mixture of these components with lysozyme despite the known instability of this enzyme. Other active ingredients that can be added to the slurry of lysozyme and co-precipitated on the core particles to enhance their antibacterial properties are complexing agents such as citric acid and EDTA, proteinogenic amino acids such as cystine, tyrosine and glycine. , Certain lytic peptides such as cecropin or sarcotoxin and citric acid,
Contains organic acids such as malic acid and lactic acid.

After manufacture, the lysozyme-containing particles can be used to sterilize and / or preserve various food products. Lysozyme has been used to store bacon, fish cakes, sausages, fresh vegetables, fruits, tofu, milk and various other dairy products. Lysozyme is used commercially to prevent spoilage [late-blowing] of aged cheese. In fact, the granular material dissolves and forms a homogeneous suspension, so that it can be easily dissolved in water or added directly to the food during formulation.

The method of practicing the invention will be further described in the following examples.

[0015]

【Example】

[0016]

Example 1 Laboratory scale experiments were conducted using a Uni-Glatt laboratory fluidized bed dryer with variable air temperature and flow rate through the fluidized bed. The device comprises a container of core material (5 1/2 inch diameter x 6 1/2 inch height) that fits against the bottom of the expansion chamber of the device.
It has an inch Wurster insert.
The bottom plate of the Wurster has holes for sending air through the fluidized bed and the holes in the center of the plate have a larger diameter than the other holes. A cylindrical hollow tube called a partition (2 3/4 inch diameter x 6 inch long) is suspended above these large diameter tubes and is An updraft that is faster than above flows through the distribution pipe. The air flow rate is based on the amount and density of the core particles, while the fluidized bed is kept in the fluidized and dry state, the particles rise inside the distribution pipe and flow into the expansion chamber, then the distribution pipe It is arranged to fall through the outside and fall back into the slower areas of the airflow. This difference in air flow rate causes a cyclic upward and downward movement of the particles. The atomizer nozzle is attached to the bottom of the distribution pipe with the tip facing upward. This arrangement keeps the atomized liquid in co-current with the motion of the coated core, and results in a smooth coating. The rate of recirculating motion of the core can be adjusted by controlling the amount of air flowing through the distribution pipe and the amount of air flowing around the outside of the distribution pipe. The droplet size of the atomized enzyme spray solution is adjusted by controlling the liquid pumping rate and the air pressure for atomization. This process can be accelerated by the countercurrent downward spray liquid without the use of a Wurster column.

The height of the Wurster insert distribution pipe is adjusted vertically 1/4 to 3/4 inch above the bottom plate. When using dense core material, the distribution pipe is provided to impart a higher linear velocity to the air stream that raises particles through the interior of the distribution pipe and to maintain a smooth circulation of the material through the atomization zone. Up to three-quarters of the outer holes of the are closed. The overall airflow is adjusted so that good flow of core through the distribution pipe is obtained and the fluidized bed outside the distribution pipe is kept in a fluidized state. The air temperature at the inlet has a maximum temperature of 7 so that the temperature of the outlet and the particles are below 50 ° C.
Adjusted to 5 ° C. Due to the reported properties of lysozyme in solution at neutral pH, these conditions were expected to be too harsh to preserve the activity of lysozyme. Surprisingly, the activity of lysozyme was essentially unchanged after granulation as shown in Example 2. Typical exit temperatures during the coating process are 25 ° C to 40 ° C. The solid content concentration of the lysozyme slurry was 15 to 25% (w / w) of the whole. Feed rates ranged from 5 ml / min to 20 ml / min. The air pressure during atomization was in the range of 1.0 to 1.5 bar. A typical dry weight gain of core particles after lysozyme coating is 10 to 20% (w / w) depending on the desired final activity.

[0018]

Example 2 a. Non-dusting antibacterial product containing lysozyme and / or chelating agent Four spray coating experiments were carried out to produce a non-dusting granular product from spray-dried lysozyme powder. Lysozyme uses the raw materials shown in Table 1,
NaC in a Uni-Grat type fluid bed dryer as described above.
Spray-coated on 1 l.

The product AMP46 can be added to enhance the binding of lysozyme to the core particles of lysozyme,
A simple formulation with an additional protein source, Maltrin 100. Another simple formulation, AMP49, contains lactose, which dilutes lysozyme to obtain a less active product. Sample AMP50 exemplifies the admixture of chelating agent and antibacterial agent sodium citrate. The experiment designated AMP51 shows the incorporation of EDTA in the lysozyme containing granules. Essentially all lysozyme activity, regardless of additional components, was recovered despite the instability recognized by those of skill in the art.

[0020]

[Table 1]sample AMP 46 AMP 49 AMP 50 AMP 51 In spray liquid Solid NaCl 1000 1000 1000 1000Lysozyme 100 100 100 100Martrin 100 11 12.5 12.5 12.5 Lactose --- 12.6 12.6 12.6 citric acid sodium −−−− 33.6−−EDTA −− −− −− 20Theoretical recovery rate% 105 98 93 105 The resulting granules were fairly uniform in appearance. Remaining
Zozyme activity dissolves particles in phosphate buffer and
Enzyme activity using a traditional lysozyme assay
Obtained by Table 1 shows that the granulated material is granular
Retained lysozyme activity during and after the oxidization process
It is shown that. Table 2 shows the lysozyme in these granules.
Is spray dried lysozyme stored under similar conditions
It shows that it is more stable.

[0021]

Table 2 at room temperature lysozyme activity (units / mg) after 24 weeks initial final specimen activity activity remaining% for percent control standard lysozyme powder 48 17 35 --- AMP-46 4.5 2. 2 53 151 AMP-49 4.2 2.6 2.6 62 177 AMP-50 3.8 2.9 76 217 AMP-51 4.4 2.7 2.7 61 174

[0022]

The above results show that the activity of lysozyme is not destroyed during any granulation process, and that this form of lysozyme is significantly more stable than the traditional spray-dried products of the prior art. , Demonstrating that the method of the present invention is useful.

The main features and aspects of the present invention are as follows.

1. Spraying an aqueous slurry of lysozyme into the reaction chamber of a fluidized bed reactor with core particles of hydratable material,
A method of making lysozyme-containing particles comprising evaporating residual water thereby leaving dry lysozyme coated on the particulate core material, thereby providing particles containing lysozyme.

2. 2. The method according to 1 above, wherein the air temperature inside the reaction chamber is 40 ° C to 60 ° C.

3. The method of claim 1 wherein the core particles comprise a material that is highly hydratable. 4. A method according to claim 3 wherein the hydratable material comprises clay, nonpareils and agglomerated potato starch, salt particles, agglomerated trisodium citrate, bentonite granules or granules, diatomaceous earth granules or sodium citrate crystals. ..

5. The method according to 1 above, wherein the longest dimension of the core particles is 150 to 2,000 μm.

6. 15 to 25% by weight of aqueous slurry
2. The method according to 1 above, which comprises lysozyme.

7. A method according to claim 1 wherein sufficient lysozyme is deposited on the surface of the core particles to give a dry weight gain of 10 to 20%.

8. 2. The method according to 1 above, wherein the slurry contains an inactive and / or antimicrobially active component other than lysozyme.

9. 9. The method according to 8 above, wherein the slurry contains an ingredient that is inert as a binder and / or an ingredient that serves as a diluent for lysozyme.

10. 9. The method according to 8 above, wherein the slurry contains an antibacterial active ingredient selected from the group of complexing agents, protein-derived amino acids, bacteriocins, lytic peptides and organic acids.

11. 11. The method according to 10 above, wherein the complexing agent is citric acid or EDTA, the proteinogenic amino acid is cystine, alanine, tyrosine or glycine, and the lytic peptide is cecropine or sarcotoxin.

An aqueous slurry of lysozyme having a concentration of 12.15 to 25% by weight is sprayed into the reaction chamber of the fluidized bed reactor in which the core particles of hydratable material are suspended, whereby on the surface of the core particles. A method of producing spherical granules having lysozyme activity, comprising depositing a layer of dry lysozyme on.

13. A lysozyme-containing granule produced by the method described in 1 above.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location A61K 7/00 L 8615-4C 9/16 R 7329-4C

Claims (3)

[Claims] 1. A dry lysozyme coated on a particulate core material by spraying an aqueous slurry of lysozyme into the reaction chamber of a fluidized bed reactor with core particles of hydratable material thereby evaporating residual water. To provide particles containing lysozyme, thereby producing particles containing lysozyme. 2. An aqueous slurry of lysozyme having a concentration of 15 to 25% by weight is sprayed into the reaction chamber of the fluidized bed reactor in which the core particles of hydratable material are suspended, whereby on the surface of the core particles. A method of producing spherical granules having lysozyme activity, comprising depositing a layer of dry lysozyme on. 3. Granules containing lysozyme produced by the method of claim 1.