JPH0576311A - Method for decomposing cholesterol in egg yolk - Google Patents

Abstract

PURPOSE:To decompose cholesterol in egg yolk in a short time at a high decomposition ratio and obtain egg yolk excellent in digestibility and absorbability by pretreating the egg yolk with a protease, etc., and then using a microorganism, etc., having the ability to decompose the cholesterol. CONSTITUTION:Egg yolk is pretreated with preferably 0.1-2wt.% one or more of proteases such as serine protease and lipases such as phospholipase or the egg yolk is treated with the one or more of the proteases and lipases and a microorganism (e.g. the genus Arthrobacter) having the ability to decompose cholesterol in the egg yolk and one or more of cholesterol decomposing enzymes are used to decompose the cholesterol in the egg yolk.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a method for degrading cholesterol in egg yolk.

[0002]

2. Description of the Related Art In recent years, since excessive intake of dietary cholesterol has been a major cause of adult diseases such as arteriosclerosis and myocardial infarction, attention has been paid to reducing intake of dietary cholesterol. Dietary cholesterol is mostly derived from animal foods, although some are derived from plant foods. Cholesterol intake from animal foods per day for Japanese is about 256 mg, of which about 140 mg is derived from eggs (equivalent to 0.7 eggs), derived from milk and other dairy products. Approximately 12 mg, those derived from meat were approximately 58 mg, and those derived from seafood were approximately 46 mg [Yasu Sato et al., "Science of Egg Cooking and Health," p241 (198).
9) (from Kogaku Shuppan)], the intake of cholesterol derived from eggs is extremely high, and most of the cholesterol in eggs is contained in egg yolk.

From this point of view, there is a tendency for people to avoid eating eggs and foods using eggs, especially egg yolk, in order to suppress the intake of cholesterol. However, the egg
Along with milk, it is a nutritious food that is rich in proteins, lipids, minerals, vitamins, etc., and because it is inexpensive, it is a food that is regrettable to avoid cholesterol intake and its effective use. Is desirable.

On the other hand, as methods for removing cholesterol in foods, cholesterol extraction methods using organic solvents, cholesterol extraction methods using supercritical gas, etc. are known, but these methods require extensive equipment. It is also disadvantageous in terms of cost. Therefore, in order to improve the above-mentioned drawbacks of the extraction method, a method of decomposing cholesterol in food by using a microorganism having cholesterol degrading ability or a cholesterol-degrading enzyme derived from it has been proposed in recent years (JP-A-57-57). No. 86257, JP-A-1
No. 229097).

[0005]

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The inventors of the present invention tried to decompose cholesterol in food according to the above-mentioned known method using a microorganism capable of degrading cholesterol or a cholesterol-degrading enzyme. Cholesterol in dairy products and fats and oils is relatively easily and quickly decomposed by the known method,
The cholesterol in egg yolk takes a very long time to decompose,
Moreover, it was found that the cholesterol reduction rate (cholesterol degradation rate) did not improve so much even after long-term degradation treatment.

[0006]

Therefore, the inventors of the present invention proceeded with research for the purpose of effectively utilizing eggs with a high nutritional value, and for a method capable of degrading cholesterol in egg yolk in a short time with a high decomposition rate. Came. As a result, in decomposing the cholesterol in egg yolk with a microorganism capable of degrading cholesterol or with a cholesterol-degrading enzyme, egg yolk is pretreated with at least one of protease and lipase, or is treated with at least one of protease and lipase. It was found that the cholesterol in egg yolk can be decomposed in a short time at a high decomposition rate by simultaneously performing the above.

Therefore, according to the present invention, the egg yolk is pretreated with at least one of protease and lipase, or the egg yolk is treated with at least one of protease and lipase, and at the same time, cholesterol in the yolk is decomposed into cholesterol. A method for decomposing cholesterol in egg yolk, which comprises decomposing it using at least one of a microorganism (hereinafter referred to as "cholesterol-degrading microorganism") and a cholesterol-decomposing enzyme.

The reason why the present invention promotes the decomposition of cholesterol in egg yolk is not clear, but it is presumed to be as follows. Most of the cholesterol in egg yolk is present in low density lipoprotein (LDL). LDL
Has a core of triglyceride in the center, and the core is surrounded by a layer composed of phospholipid, protein and cholesterol, and cholesterol is mixed with phospholipid and protein. Therefore, cholesterol in egg yolk is in a state of being extremely unlikely to be affected by cholesterol-degrading microorganisms and cholesterol-degrading enzymes, and when directly acting on cholesterol-degrading microorganisms and degrading enzymes, it is blocked by phospholipids and proteins. Cholesterol is not decomposed smoothly. On the other hand, in the present invention, the protease and / or lipase decomposes phospholipids and proteins around cholesterol, which facilitates the action of cholesterol-degrading microorganisms and cholesterol-degrading enzymes on cholesterol, thus reducing cholesterol in a short time. In addition, it is considered that it will be decomposed at a high decomposition rate.

In the present invention, egg yolk may be treated with only one of protease and lipase, or both enzymes may be used to treat egg yolk. If both enzymes are used, the yolk should be treated with a protease followed by a lipase, with a lipase followed by a protease, or both lipase and a protease if they do not interfere with each other's action. May be used for simultaneous processing. Only one type of protease may be used or two or more types may be used, and the same applies to lipase.

The treatment with the protease and / or lipase may be carried out before the egg yolk is treated with the cholesterol-degrading microorganism and / or the cholesterol-degrading enzyme, or the protease and lipase may interfere with the action of the cholesterol-degrading microorganism and the cholesterol-degrading enzyme. If not, it may be performed at the same time as the cholesterol decomposition treatment. Usually, it is preferable to treat the egg yolk with at least one of protease and lipase and then to decompose the cholesterol in the egg yolk because the decomposition rate of cholesterol can be further increased.

The method of the present invention can be carried out directly on whole eggs, or directly on the yolk after separating the yolk from the egg, using a liquid obtained by diluting the yolk with a liquid medium such as water. Alternatively, it may be performed on a food product containing egg yolk if it does not adversely affect other ingredients in the food product. In particular, when the yolk to be subjected to the decomposition treatment is the yolk itself or high-concentration yolk liquid, cholesterol in the yolk is hardly decomposed by the conventional technique of only treating with cholesterol-degrading microorganisms or cholesterol-degrading enzymes, but protease and lipase In the present invention in which the treatment with at least one of the above is performed in combination with the cholesterol decomposition treatment, cholesterol in egg yolk can be decomposed in a short time at a high decomposition rate.

[0012] Further, the protease and lipase may be used in a free state, or may be immobilized in a suitable solid medium or the like by any method and then used. Further, the treatment with these enzymes may be performed by a batch method or a continuous method.

As the protease, various proteases can be used, for example, serine protease (trypsin, chymotrypsin, thrombin, plasmin, elastase etc.), cysteine protease (papain, ficin, bromelain etc.), aspartic protease (pepsin, Cathepsin, renin, etc.), metalloproteases and the like. Of these, serine protease is particularly preferable.

As the lipase, lipase, phospholipase, lipoprotein lipase and the like can be used, and specific examples thereof include triacylglycerol lipase, phospholipase A ₁ , A ₂ , C and D.
Of these, phospholipase C is particularly preferable.

LDL containing cholesterol in egg yolk
In addition to the lipids and proteins present in it, it also contains large amounts of other lipids and proteins. Therefore, when it is desired to suppress the decomposition of lipids and proteins other than LDL, it is preferable to select and use a lipase or protease that selectively acts on the lipids and proteins in LDL. On the other hand, lipases and / or proteases that act on both lipids and proteins in LDL and on non-LDL lipids and proteins can also be used. In that case, at the same time that the lipids and proteins contained in the LDL in the yolk are degraded, the LD in the yolk
Since a large amount of other lipids and proteins contained in addition to L can be decomposed and converted into a low molecular weight compound that is easily digested and absorbed, it is possible to obtain an egg yolk or an egg yolk product having excellent digestion and absorption.

The proportion of protease or lipase used varies depending on various requirements such as the type of egg yolk or egg yolk liquid to be treated, the type and activity of the enzyme, the treatment conditions, etc., but it is usually about 0 based on the weight of the egg yolk. 0.01-5% (about 10-50
00 unit / egg yolk 1), more preferably about 0.1-
2% (about 100 to 2000 units / gum yolk 1 g) is preferable. When the treatment with at least one of protease and lipase is carried out before the cholesterol decomposition treatment, it is usually at an optimum pH and an optimum temperature of the protease and lipase to be used, and usually about 1 to 48 hours, preferably about 4 to Pretreatment with at least one of 12 hours protease and lipase is preferred. When the treatment with at least one of protease and lipase is performed at the same time as the cholesterol decomposition treatment, it is usually about 12 to
The simultaneous treatment may be performed for 240 hours, preferably about 24 to 180 hours.

The cholesterol-degrading microorganisms include genus Arthrobacter, genus Protaminobacterium, genus Microbacterium, genus Prepibacterium, genus Corynebacterium, genus Bacillus, genus Nocardia, genus Serratia,
Any of microorganisms such as Mycobacterium, Streptomyces, Rhodococcus, and other known cholesterol-degrading microorganisms can be used.

Specifically, Arthrobacter simplex (IAM-1660), Protaminobacterium arboflaps (IAM-1040), Microbacterium lacticum (IAM-1640), Prepibacterium lipolyticum (IAM) -1398), Corynebacterium equi (IAM-1038), Bacillus roseumii (IAM-1257), Bacillus sphaericus (ATCC-7055), Nocardia coralina (I).
FO-3338), Nocardia Minima (IAM-03)
74), Nocoalzia Cardoneri (IAM-010)
5), Serratia marcescens (IAM-125
5), Mycobacterium frey (IFO-315
8), Mycobacterium avium (IFO-308
3), Streptomyces spescens (IAM-00
74), Rhodococcus equi (JCM-6819),
Rhodococcus erythro hollis (JCM-6824),
Examples include microorganisms such as Rhodococcus rhodochrous (JCM-3202) and Rhodococcus coralinas (JCM-3199), and all of the above microorganisms are known.

When the cholesterol-degrading microorganism is used to decompose cholesterol, it is preferable to add a nutrient source for the microorganism such as yeast extract, because the decomposition of cholesterol can be further promoted. Further, as the cholesterol-degrading enzyme, any of the cholesterol-degrading enzymes produced by culturing the above-mentioned cholesterol-degrading microorganisms and other known cholesterol-degrading enzymes can be used.

The cholesterol-degrading microorganism and the cholesterol-degrading enzyme may be used alone or in combination. In addition, two or more kinds of cholesterol-decomposing microorganisms and / or cholesterol-decomposing enzymes can be used in combination when the microorganisms or the enzymes do not interfere with each other.

The above-mentioned microorganisms capable of degrading cholesterol are disclosed in, for example, JP-A-57-86257 and JP-A-1.
No. 229097, any known method (for example, a method of preparing a preculture solution, a method of centrifuging a supernatant to obtain an enzyme solution, a method of crushing separated bacterial cells,
It can be prepared by a method such as a method of preparing an extract having a resolution, and this may be applied to egg yolk.

When degrading cholesterol, the above-mentioned cholesterol-degrading microorganism and cholesterol-degrading enzyme may be used in a free state, or may be immobilized on a suitable solid medium or the like by any method. .. Further, the cholesterol decomposition treatment may be performed by a batch method or a continuous method. The whole egg, egg yolk or egg yolk liquid obtained by the treatment of the present invention can be used as it is, or after being concentrated or diluted or subjected to other treatment, for use in foods and drinks and other uses.

The present invention will be specifically described below with reference to examples and the like, but the present invention is not limited thereto. In the following examples,% means% by weight. The cholesterol content was measured as follows.

[Measurement of Cholesterol Content] Sample Solution 2
8 ml of isopropyl alcohol is added to ml
Shake for 10 minutes at warm to perform extraction process, then
High-performance liquid chromatographic analysis of Ropil alcohol solution under the following conditions:
Cholestero in sample solution after graph (HPLC) treatment
Content was measured.HPLC conditions  Column: LQ PACK Nucleosil Mobile phase: Methanol Liquid speed: 1 ml / min Detection: UV210nm

Example 1 Cholesterol-degrading microorganisms Rhodococcus equi (JCM-6819) (RIKEN) were used, NH ₄ NO ₃ 0.1%, K ₂ HPO ₄ 0.0025.
_{%, MgSO 4 · 7H 2 O} 0.0025%, FeSO 4 · 7H 2 O 0.000
10 ml of medium containing 01%, yeast extract 0.5%, cholesterol 0.1% and Tween 20 0.1% (pH 7.0)
Was inoculated into. After inoculation, shaking culture was performed at 30 ° C. for 24 hours while rotating at a rotating speed of 90 times / min. Do this operation 2
Precultures were prepared by repeating ~ 3 times. After preparing a 10% egg yolk aqueous solution containing 0.5% yeast extract, 300 mg of Protin FN (protease manufactured by Daiwa Kasei Co., Ltd.) was added to 100 ml of this egg yolk aqueous solution, and the mixture was added at 45 ° C. for 1 hour.
Treated for 2 hours. Next, after heating at 65 ° C for 15 minutes to inactivate the enzyme, 2 ml of the preculture liquid of Rhodococcus spp. Prepared above was added to this liquid, and the mixture was kept at 30 ° C for 48 hours for 2 hours.
The cholesterol was decomposed by rotating and shaking at a rotation speed of 00 rpm / min. The cholesterol contents in the egg yolk aqueous solution before the cholesterol decomposition treatment (0 hour), after 24 hours and after 48 hours were measured by the above-mentioned method and were as shown in Table 1 below.

Comparative Example 1 Cholesterol was decomposed in the same manner as in Example 1 except that pretreatment with Protin FN was not carried out. As a result, cholesterol in the yolk aqueous solution after 0 hour, 24 hours and 48 hours was obtained. The content was as shown in Table 1. In Table 1, the cholesterol decomposition rate was calculated by the following formula.

Cholesterol decomposition rate (%) = {(A ₀ −
A ₁ ) / A ₀ } × 100 A ₀ : Cholesterol content (mg / ml) of egg yolk aqueous solution before cholesterol decomposition treatment (0 hours) A ₁ : Egg yolk 24 hours, 48 hours or 120 hours after cholesterol decomposition treatment Cholesterol content of aqueous solution (mg / ml)

[0028]

[Table 1]  Example 1 Comparative example 1  0 hours  Cholesterol content (mg / ml) 0.153 0.144 Cholesterol degradation rate (%) 0.0 0.024 hours later  Cholesterol content (mg / ml) 0.077 0.143 Cholesterol degradation rate (%) 49.7 0.748 hours later  Cholesterol content (mg / ml) 0.006 0.138 Cholesterol degradation rate (%) 96.1 4.2

From the results shown in Table 1, in Example 1 of the present invention in which the protease treatment was carried out before the cholesterol decomposition treatment, the decomposition rate of cholesterol reached about 50% after 24 hours and about 96% after 48 hours. It can be seen that the cholesterol in egg yolk is decomposed in a short time and at a high decomposition rate. On the other hand, Comparative Example 1 in which cholesterol decomposition treatment is performed without treatment with protease
Then, the degradation rate of cholesterol was only 0.7% after 24 hours, and was only 4.2% after 48 hours,
It can be seen that cholesterol in egg yolk is not decomposed even after long-term decomposition treatment.

Example 2 In the same manner as in Example 1, a preculture liquid containing Rhodococcus sp. Was prepared. After preparing a 50% egg yolk aqueous solution containing 0.5% of yeast extract, 500 mg of Protin FN was added to 100 ml of this egg yolk aqueous solution and treated at 45 ° C. for 12 hours. Then at 65 ° C for 15
After heating for a minute to deactivate the enzyme, 2 ml of the preculture liquid of Rhodococcus spp.
Cholesterol was decomposed by rotating and shaking at 200 ° C. for 120 hours at a rotation speed of 200 rpm / min. Before (0 hours), 48 hours and 1
The cholesterol content in the yolk aqueous solution after 20 hours was measured by the above-mentioned measuring method, and was as shown in Table 2 below.

Comparative Example 2 When cholesterol was decomposed in the same manner as in Example 2 except that pretreatment with Protin FN was not performed, cholesterol in the yolk aqueous solution after 0 hour, 48 hours and 120 hours was measured. The content was as shown in Table 2.

[0032]

[Table 2]  Example 2 Comparative example 2  0 hours  Cholesterol content (mg / ml) 0.780 0.718 Cholesterol degradation rate (%) 0.0 0.048 hours later  Cholesterol content (mg / ml) 0.692 0.709 Cholesterol degradation rate (%) 11.3 1.3120 hours later  Cholesterol content (mg / ml) 0.117 0.532 Cholesterol degradation rate (%) 85.0 25.9

From the results shown in Table 2 above, in Example 2 of the present invention in which the protease treatment was performed before the cholesterol decomposition treatment, the cholesterol decomposition rate reached about 85% after 120 hours, and the cholesterol in egg yolk was increased. In contrast to the high decomposition rate, in Comparative Example 2 in which cholesterol is decomposed without treatment with protease, the decomposition rate of cholesterol is about 26% after 120 hours.
It is understood that cholesterol in egg yolk is not decomposed at a high rate even after the decomposition treatment for a long time.

Example 3 A preculture liquid containing Rhodococcus was prepared in the same manner as in Example 1. After preparing a 50% egg yolk aqueous solution containing 0.5% yeast extract, 100 μl of paratase M (lipase manufactured by Novo Industry Co .; triacylglycerol lipase) was added to 100 ml of this egg yolk aqueous solution and treated at 45 ° C. for 12 hours. ..
Then heat at 65 ° C for 15 minutes to inactivate the enzyme,
2 ml of the preculture liquid of Rhodococcus spp. Prepared above was added, and cholesterol was decomposed by shaking at 30 ° C. for 48 hours at a rotation speed of 200 rpm / min. The cholesterol content in the egg yolk aqueous solution before the cholesterol decomposition treatment (0 hour), after 24 hours and after 48 hours was measured by the above-mentioned measuring method, and it was as shown in Table 3 below.

Comparative Example 3 When cholesterol was decomposed in the same manner as in Example 2 except that pretreatment with Patalase M was not carried out, cholesterol in the yolk aqueous solution after 0 hour, 24 hours and 48 hours was measured. The content was as shown in Table 3.

[0036]

[Table 3]  Example 3 Comparative example 3  0 hours  Cholesterol content (mg / ml) 0.139 0.144 Cholesterol degradation rate (%) 0.0 0.024 hours later  Cholesterol content (mg / ml) 0.104 0.143 Cholesterol degradation rate (%) 25.2 0.748 hours later  Cholesterol content (mg / ml) 0.080 0.138 Cholesterol degradation rate (%) 42.4 4.2

From the results of Table 3 above, in Example 3 of the present invention in which lipase treatment was performed before cholesterol decomposition treatment, the decomposition rate of cholesterol reached about 25% after 24 hours and about 42% after 48 hours. Cholesterol in egg yolk is decomposed at a high decomposition rate in a short time, whereas in Comparative Example 3 in which cholesterol decomposition treatment is performed without treatment with lipase, the decomposition rate of cholesterol is 24 hours. After that, it was only 0.7%, and even after 48 hours it was only 4.2%, which shows that cholesterol in egg yolk is not decomposed even after a long-time decomposition treatment.

Example 4 2.5 g of yeast extract and 3 g of Protin FN were added to 500 ml of egg yolk to give 45
It was treated at ° C for 12 hours. Then, after heating at 65 ° C for 15 minutes to inactivate the enzyme, 10 ml of the same preculture liquid of Rhodococcus as used in Example 1 was added, and the rotation speed was 200 rpm / min at 30 ° C for 120 hours. The mixture was rotatably shaken to decompose cholesterol. As a result, the cholesterol content in egg yolk was 1.423 mg / ml before the treatment with Rhodococcus, but after treatment it was 0.43 mg / ml.
It was reduced to 241 mg / ml, and the decomposition rate of cholesterol was as high as 83.1%. From this, it can be seen that cholesterol in egg yolk is decomposed extremely efficiently by the method of the present invention in which the treatment is performed with a protease and then the cholesterol is decomposed.

[0039]

According to the method of the present invention, cholesterol in egg yolk can be decomposed at a high decomposition rate in a short time. And, since the egg yolk obtained by the method of the present invention and the product containing it have a very low cholesterol content, it is possible to effectively ingest other nutrients while keeping the intake of cholesterol low when eating or drinking. ..
Furthermore, in the present invention, when an enzyme that can decompose not only proteins and lipids in LDL containing cholesterol but also other proteins and lipids contained in egg yolk is selected and used as the protease and lipase,
Since proteins and lipids other than LDL of egg yolk are decomposed and converted into low molecular weight compounds that are easily digested and absorbed, it is possible to provide whole egg, egg yolk, or an egg yolk-containing product having excellent digestion and absorption properties.

Claims (1)

[Claims] 1. After pretreatment of egg yolk with at least one of protease and lipase, or at the same time when egg yolk is treated with at least one of protease and lipase, cholesterol in egg yolk of microorganisms having cholesterol degrading ability and cholesterol degrading enzyme A method for decomposing cholesterol in egg yolk, which comprises decomposing using at least one kind.