CN103315203A - Royal jelly solution and its preparation method - Google Patents

Abstract

A royal jelly solution and a method for producing the same, the method comprising: a royal jelly raw material is mixed with water to obtain a royal jelly aqueous suspension, and the royal jelly aqueous suspension is added with an enzyme containing cysteine endonuclease and then reacted at a temperature of 20 to 70 ℃ to obtain a royal jelly solution which is in a coagulated and stable suspension state. By adding the ferment containing cysteine endonuclease into the royal jelly water suspension, the hydration capability of protein molecules in the royal jelly solution can be improved, so that the protein molecules in the royal jelly solution are not easy to separate and precipitate and are in a stable suspension state, and centrifugation, filtration, emulsification and homogenization treatment are not needed, so that the whole components of the royal jelly can be reserved, the nutritive value is higher, the treatment procedure can be reduced, and the process efficiency and the economic benefit are better.

Description

Royal jelly solution and manufacturing method thereof

technical field

The present invention relates to a royal jelly solution and a manufacturing method thereof, in particular to a biotechnological special fermentation method for treating raw royal jelly to make it present as a non-transparent curd, that is, using this biotechnological enzyme treatment technology to make royal jelly The royal jelly solution which is in the form of stable suspension and opalescent and non-transparent and a preparation method thereof.

Background technique

Royal jelly, also known as royal jelly, is a yellow-white viscous slurry secreted by the throat glands of worker bees. Studies have found that larvae fed royal jelly throughout the larval stage can develop into queen bees in only 16 days, and their life span is as long as 5-6 years. Therefore, royal jelly is considered to have the effect of prolonging life.

Generally, fresh royal jelly is milky white or light yellow. It is a paste-like semi-fluid, partially soluble in water and ethanol, and will produce white precipitates. After a period of time, there will be stratification. The water content of royal jelly is 60-70% on average, and contains 9-18% protein, 7-16% carbohydrate and 3-8% lipid, other components also have free amino acid, vitamin (for example, Biotin, folic acid, inositol, niacin, pantothenic acid, vitamin B ₁ , B ₂ , vitamin E), minerals, peptides, fatty acids and bioactive substances that have health care functions for the human body, such as decenoic acid (10 -Hydroxy-2-decenoic acid, referred to as 10-HDA) and MRJPs (Major Royal Jelly Proteins), etc. Therefore, royal jelly is also often used as a natural nutritional health product, and in the application case of Taiwan Patent Publication No. 200526193, it is also disclosed that royal jelly is used as one of the ingredients of the composition for anti-aging, Taiwan Patent of China Publication No. I340651 patent case reveals that royal jelly can be used to enhance collagen production.

Although the original royal jelly can be eaten directly, it is not easy to be accepted by the public because of its special astringent pungent, sour and sticky taste, and low affinity to the throat. In addition, the original royal jelly needs to be stored in freezing or cold storage, mainly because the protein and proteases contained in royal jelly are sensitive to temperature, and royal jelly is prone to browning in high-temperature storage environments, so its preservation is not good. It is better and affects the convenience of eating. In order to overcome the problem that it is not easy to preserve, the food industry usually processes royal jelly into powder and fills it into capsules or lozenges. Although the No. CN100421578 patent case of Chinese Patent Announcement, the No. CN101223946 patent case of Chinese Patent Announcement, the No. CN1076362A application case of Chinese Patent Publication, and the Japanese Patent Publication No. 5-9125 application case disclose respectively the method that royal jelly is made into beverage , and improve the preservation of the royal jelly solution by mixing it with a large amount of sugar and colloid or making it into a dry powder, but the aforementioned treatment method can only mix a small amount of royal jelly into liquid foods such as beverages, and the original queen bee The amount of milk used in processed beverages is still limited, and the nutritional value of royal jelly-related drinks is still unsatisfactory. Therefore, there is still a need to develop processing technologies that can increase the amount of royal jelly added.

Among them, due to the unique rheological properties of royal jelly, it is easy to form a pseudoplastic body when the temperature is greater than 25 ° C, and cause difficulty in quality control during the process and storage. Although the Chinese Patent Application No. CN1076362A and Japan Patent Application No. 5-320060 discloses that after dissolving royal jelly in water, first centrifuge to remove the non-water-soluble sediment, and then extract the active ingredients in the sediment with ethanol or acetone, and then heat and adjust its pH After the value is reached, the precipitate is processed by centrifugation or membrane filtration to obtain an extract containing active ingredients, and the extract is added back to the royal jelly aqueous solution that removes the precipitate, and thereby removes the royal jelly The insoluble components of pseudoplastics, but this treatment method is easy to remove the alcohol-insoluble protein in royal jelly at the same time, but cannot completely retain the active ingredients in royal jelly, and the solvent used to extract the active ingredients in the sediment remains in the final product May be harmful to the health of consumers. And Japanese Patent Application No. Hei 1-215268 and Japanese Patent Application No. 2008-118869 disclose the content of adding preservatives in royal jelly aqueous solution to improve its preservation. However, adding preservatives still has the potential to endanger the health of consumers. risks of.

In addition, Taiwan Patent No. 208043 and Japanese Patent Application No. Hei 2-333641 respectively disclose the method of enzymatically decomposing protein to improve the protein decomposition rate, recovery rate and preservation of royal jelly, and then obtain transparent and clear The contents of the royal jelly solution. However, the above-mentioned methods all need to use more than two kinds of proteases to act on the royal jelly solution with adjusted pH value, and there are still a large amount of insoluble protein precipitates to form obvious stratification. In addition, the product is preserved due to excessive precipitates. Due to the limited performance and poor appearance, consumers may doubt the quality of the product. Therefore, centrifugal filtration or membrane filtration is usually used to remove the sediment to obtain a clear royal jelly solution without obvious sediment. However, after centrifugation or filtration, the unique active (bioactive) substances or insoluble proteins in royal jelly will be removed due to centrifugation or filtration, resulting in the incomplete retention of all proteins and active ingredients in royal jelly, and some The disadvantage of relatively poor nutritional value. Moreover, the technical content disclosed in the aforementioned application obviously needs to use more than two kinds of enzymes from mammals to treat the royal jelly solution to achieve a higher recovery rate. Therefore, the above-mentioned existing method has the defect that the production cost is higher and does not meet the economic benefit because of using more than two kinds of enzymes from mammals and filtering to remove the sediment procedure. In addition, in the existing production methods, the original royal jelly is still prone to white turbidity or coagulation precipitation due to excessive addition, which cannot maintain a stable suspension state, and makes the addition amount limited, resulting in the produced There is still room for improvement in the nutritional value of royal jelly drinks.

Therefore, royal jelly with good stability and high nutritional value is still being developed to meet the needs of the food market. In addition, in order to reduce the waste of royal jelly due to processing, there are also methods for improving royal jelly solution-related products. demand.

Contents of the invention

The purpose of the present invention is to provide a biotechnology enzyme treatment containing a specific enzyme that can be in a stable suspension state without obvious sedimentation and stratification, so that it does not need to carry out a treatment procedure to remove the sediment and can retain the integrity A method for producing a royal jelly solution containing royal jelly nutrients.

The manufacture method of royal jelly solution of the present invention comprises the following steps:

One, mixing a royal jelly raw material with water to obtain a royal jelly aqueous suspension;

2. Adding an enzyme containing cysteine endopeptidase to the royal jelly aqueous suspension to obtain a royal jelly pre-reaction solution; and

3. Reacting the royal jelly pre-reaction solution at a temperature of 20° C. to 70° C. to obtain a curd-like royal jelly solution in a stable suspension state.

The beneficial effect of the manufacture method of royal jelly solution of the present invention is: the protein of royal jelly is mainly made up of water-soluble and water-insoluble protein, and wherein royal jelly protein (MRJPs) accounts for about 90% of total water-soluble protein content, and in MRJPs protein there is It has a cysteine site and its C-terminus has a highly hydrophobic property, so this method can degrade royal jelly protein into Peptide or amino acid molecules with smaller molecules, and the ionizable amino and carboxyl groups are revealed with hydrolysis. In addition, endocysteine can also act on royal jelly protein, especially the characteristics of thiol groups (-SH, thiol) in proteins, and can pass through the hydrogen bonds formed between thiol groups, To improve the hydration capacity of the protein molecules in the royal jelly solution, through the degradation of the protein and the characteristics of the endocysteine enzyme acting on specific groups, the protein molecules in the prepared royal jelly solution are not easy to precipitate and can It maintains a stable suspension state for a long time, so it is not necessary to remove the sediment by centrifugation or filtration as in the existing method. Therefore, the production method of the present invention has the ability to retain the entire composition of royal jelly to provide a royal jelly solution product with higher nutritional value characteristics. In addition, since the royal jelly solution prepared by the manufacturing method of the present invention can maintain a stable suspension state, the royal jelly solution can be used as a raw material for beverages or liquid preparations without further filtration, emulsification, and homogenization, which can reduce the amount of processing The process has better process efficiency and economic benefits.

Further, the present invention also provides a royal jelly solution that does not form obvious stratification and can maintain a stable suspension state.

The royal jelly solution of the present invention is obtained by adding an enzyme containing endocysteine to a royal jelly aqueous suspension formed by mixing a royal jelly raw material with water, and the royal jelly solution is curd-like It is in a stable suspension state and contains proteins with the following characteristic protein masses: 1214 m/z ± 0.5% and/or 2032 m/z ± 0.5%.

The beneficial effects of the royal jelly solution of the present invention are: in the royal jelly solution prepared by adding an enzyme containing endocysteine to the royal jelly aqueous suspension, in addition to the above-mentioned characteristics appearing in the royal jelly solution In addition to the quality of the protein, it is also in the form of curd and stable suspension, and there will be no obvious sedimentation and stratification without centrifugation or filtration, so that the royal jelly solution can be kept in a complete royal jelly solution. Under the conditions, the whole royal milk drink or liquid product with complete nutritional ingredients can be directly made, and has the characteristics of higher nutritional value.

Description of drawings

Fig. 1 is a flow chart of a preferred embodiment of the manufacture method of royal jelly solution of the present invention;

Figure 2 is a comparison diagram of the royal jelly water suspension in the states of sedimentation, stratification and suspension after different treatment methods;

Fig. 3 is a bar graph, is used for contrasting the stability coefficient of the test solution of different ferment treatments;

Fig. 4 is a mass spectrogram, is used for illustrating the protein mass distribution situation in test solution D1;

Fig. 5 is a mass spectrogram, is used for illustrating the protein mass distribution situation in test solution D2;

Fig. 6 is a mass spectrogram, is used for illustrating the protein mass distribution situation in test solution D3;

Fig. 7 is a mass spectrogram, is used for illustrating the protein mass distribution situation in test solution D4;

Fig. 8 is a mass spectrogram, is used for illustrating the protein mass distribution situation in test solution D5;

Fig. 9 is a mass spectrogram, is used for illustrating the protein mass distribution situation in test solution D6;

Fig. 10 is a mass spectrogram for illustrating the mass distribution of the protein in the test solution D7.

Detailed ways

In order to make the above objects, features and advantages of the present invention more comprehensible, specific implementations of the present invention will be described in detail below in conjunction with the accompanying drawings. First of all, it should be noted that the present invention is not limited to the following specific embodiments. Those skilled in the art should understand the present invention from the spirit embodied in the following embodiments, and each technical term can be optimized based on the spirit of the present invention. broad understanding.

Referring to Fig. 1, a preferred embodiment of the manufacture method of royal jelly solution of the present invention comprises the following steps:

Step 201 is to mix a royal jelly raw material with water to obtain an aqueous royal jelly suspension. Wherein, the royal jelly raw material can be selected from original raw royal jelly, frozen royal jelly or freeze-dried royal jelly. The water temperature for preparing the aqueous suspension of royal jelly is not limited. However, in order to quickly complete the preparation and avoid browning of the protein in the royal jelly raw material caused by excessive water temperature, it is preferable to prepare the royal jelly aqueous suspension at a water temperature of 20°C to 70°C, more preferably at a temperature of 40°C to 55°C. ℃ water temperature preparation. In addition, the concentration of royal jelly raw materials in the royal jelly aqueous suspension is not limited, and the concentration of royal jelly raw materials in the royal jelly aqueous suspension can be adjusted according to requirements. The royal jelly raw materials in the royal jelly aqueous suspension The concentration is preferably 0.1-40% by weight (wt%), more preferably 2-20wt%.

Step 202 is to add an enzyme containing cysteine endopeptidase to the royal jelly aqueous suspension to obtain a royal jelly pre-reaction liquid. The ferment used in the present embodiment is the papaya ferment (papain) that comes from plant or other ways to make (in this embodiment, select the papaya ferment that EC number is 3.4.22.2, 3.4.22.6 and 3.4.22.30), pineapple Ferment (bromelain) (in the present embodiment, select the pineapple ferment that EC numbering is 3.4.22.32 and 3.4.22.33), fig ferment (ficain) (in the present embodiment, select the fig ferment that EC numbering is 3.4.22.3) , or a combination of the aforementioned enzymes. Wherein, the desired effect can be obtained only by adding one of the aforementioned enzymes, and the added amount of the enzyme can be 0.01% to 90% of the weight of the royal jelly aqueous suspension. Preferably, in order to avoid flocculation caused by too high dosage of the enzyme, the added amount of the enzyme is 0.05%-10%, more preferably 0.1%-5%, of the weight of the royal jelly aqueous suspension.

Among them, the above-mentioned EC number is a set of numbering classification method for enzymes (enzymes) made by the Enzyme Commission based on the chemical reaction catalyzed by each enzyme. According to this, each enzyme is assigned a The recommended serial number, each enzyme serial number starts with the letter "EC", and then is represented by four numbers, the number at the rear position represents a finer classification, so that the classification and properties of the enzyme can be seen through the EC serial number . For example, the number of tripeptide aminoprotease is EC3.4.11.4, where "EC3" refers to the hydrolase, "EC3.4" refers to the hydrolase acting on the peptide bond, and "EC3.4.11" refers to the peptide "EC3.4.11.4" is a hydrolase that separates the amino terminus of tripeptides.

Preferably, since the ferment containing endocysteine used in the present invention, for example, papain, pineapple ferment and fig ferment are all acid-neutral proteases, in order to obtain better reaction efficiency, the queen bee can be prepared in advance The pH value of the milk-water suspension is adjusted to a range suitable for the action of the above-mentioned enzymes. Since the pH value of the aqueous royal jelly suspension prepared in step 201 is usually in the range of 3-5, the pH value of the aqueous royal jelly suspension may not be adjusted, and enzymes may be directly added for reaction. In addition, when the pH value of the royal jelly aqueous suspension changes due to different sources of royal jelly raw materials, or different batches of raw materials, or the concentration of royal jelly in the royal jelly aqueous suspension, it can also be used in the food industry or The pH value adjustment of the royal jelly aqueous suspension is carried out by a pH value regulator that is medically approved and meets safety specifications. Commonly used regulators are: citric acid, tartaric acid, malic acid, sodium hydroxide, sodium bicarbonate, citric acid and lactic acid. You can use the above regulator to adjust the pH value of the royal jelly aqueous suspension to 2-8 first, and then add enzymes. If you want to get better reaction efficiency, it is better to adjust the pH value of the royal jelly aqueous suspension 3-6, more preferably 3-5.

Step 203 is to make the royal jelly pre-reaction liquid react at a temperature of 20°C-70°C for about 0.5-10 hours, and then obtain a curd-like royal jelly with almost no precipitation and no layering and a stable suspension state solution. Among them, in order to avoid browning under high temperature and cause the royal jelly solution to be unstable, the enzyme reaction temperature is preferably between 30-60°C, more preferably 35-55°C.

In order to achieve the required reaction effect and avoid the reaction time being too long, which does not meet the requirements of manufacturing efficiency, the reaction time can be further adjusted by adjusting the amount of enzyme added and the reaction temperature. It is better to pre-react the royal jelly The reaction time of the solution is controlled at 0.6 to 5 hours, more preferably 0.7 to 3 hours. When the required reaction effect or the set reaction time is reached, a method selected from the following groups can be used to inhibit the enzyme from continuing Function: heating the royal jelly pre-reaction liquid to a temperature of 70°C to 110°C, or adjusting the pH of the royal jelly pre-reaction liquid to less than 2 or greater than 8. That is, the enzyme is inactivated by high temperature and a pH environment that is not suitable for the activity of this type of enzyme and stops its function. In this embodiment, heating to a high temperature is used to inhibit the continued action of the enzyme. Among them, in order to avoid browning of the royal jelly solution after being treated with papaya enzyme or pineapple enzyme, the temperature for inhibiting enzyme activity is preferably set within the range of 80-105°C, more preferably within the range of 90-100°C.

It is worth mentioning that when the royal jelly solution obtained in this step is used for mass spectrometry analysis of proteins, it can be found that the royal jelly solution obtained by the method of the present invention contains proteins with the following characteristic protein quality: 1016m/z± 0.5%, 1214m/z±0.5%, 2016m/z±0.5%, 2032m/z±0.5%, 3477m/z±0.5%, 6030m/z±0.5%, 6314m/z±0.5%, 7413m/z±0.5 % and 8640m/z±0.5%. Wherein, after the above-mentioned enzyme treatment, at least one of the proteins whose characteristic protein mass is 1214m/z±0.5% and 2032m/z±0.5% will exist in the royal jelly solution, and the rest of the protein mass may all exist. It may also be only partially present.

It should be added that in step 202, only one of the above-mentioned enzymes needs to be added to the royal jelly aqueous suspension, and the effect of endocysteine can be utilized without adding emulsifiers or stabilizers. , excipients, anticoagulants, dissolution aids, stabilizers, preservatives, thickeners and other additives allowed in the general food industry, the obtained non-transparent, non-clear water curd-like, almost Suspended, non-settling, non-layered and stable royal jelly solution. Since the obtained royal jelly solution is in a stable suspension state, it does not need to be clarified or filtered, so that the royal jelly solution can retain the complete royal jelly components In addition, the stability of the royal jelly solution after the above-mentioned enzyme treatment is good, and it still has the characteristics of long-term preservation without adding stabilizers and preservatives, so that the royal jelly solution of the present invention can retain complete nutrients It has better nutritional value without adding food additives. In addition, compared with the previous enzyme treatment method, two or more protein enzymes are usually added to the royal jelly aqueous suspension to obtain the required treatment effect. The present invention only needs to add one kind of enzyme, and the way of using plant enzyme can save the cost of purchasing enzyme, and at the same time, it is more economical and environmentally friendly.

In addition, based on the consideration of product diversification development and design, the royal jelly solution can be further prepared into beverage products with different forms, different edible characteristics, different flavors or other additional effects by matching additives approved by medicine or food regulations. For example, medically or food-approved additives and materials such as nutrients, health food ingredients, health food ingredients, excipients, stabilizers, thickeners, and anticoagulants can be added to the royal jelly solution. For example, medical or food-approved additives or flavorings can be added to the royal jelly solution to further produce a royal jelly beverage product containing 40 g of the aforementioned royal jelly solution per 100 cc.

The royal jelly solution of the present invention is prepared by the aforementioned method, that is, the royal jelly solution is to add an enzyme containing endocysteine to a royal jelly aqueous suspension formed by mixing a royal jelly raw material with water Obtained after the reaction, the royal jelly solution is curd-like and in a stable suspension state, its pH value is 3-5, and contains proteins with the following characteristic protein qualities: 1214m/z±0.5% and/or 2032m/z±0.5% . Further, in addition to at least one of the above two main characteristic protein qualities will exist in the royal jelly solution, the royal jelly solution may also contain proteins with at least one of the following characteristic protein qualities: 1016m/z±0.5%, 2016m/z±0.5%, 3477m/z±0.5%, 6030m/z±0.5%, 6314m/z±0.5%, 7413m/z±0.5%, and 8640m/z±0.5%.

Wherein, the enzyme containing endocysteine can be papaya enzyme, pineapple enzyme, fig enzyme, or a combination of the aforementioned enzymes, and its added amount can be 0.01% to 90% of the weight of the royal jelly aqueous suspension, Preferably it is 0.05% to 10%. Since the aforementioned enzymes all belong to endocysteine enzymes in the classification of enzymes, they can all act on thiol groups (thiol, -SH) in proteins. After being acted on by any of the aforementioned three enzymes, the queen bee Milk protein will be degraded into peptides or smaller amino acid molecules, so that the ionizable amino and carboxyl groups will be exposed with hydrolysis, and the surface charge and molecular structure of the protein will change, especially the hydrophilic groups (such as thiol group), and the hydrogen bond formed between the thiol groups also makes the hydration ability of the protein in the royal jelly solution improved due to the hydrogen bond between the groups, thus making the royal jelly solution after the action of the aforementioned enzyme The formed protein has good hydration ability and can maintain a stable suspension state without precipitation and stratification. In addition, the aforementioned three enzymes are all acid-neutral proteases, and the prepared royal jelly aqueous suspension itself is also an acid-neutral solution (its pH value is about 4). Acidity, whereby the pH value of the royal jelly solution is lower than the isoelectric point (pI) of the protein, thereby promoting the overall solution to be hydrated. Therefore, all proteins in the royal jelly solution (including insoluble proteins that are easy to form precipitates), after adding an enzyme containing endocysteine, coordinate pH value, protein isoelectric point and hydrophilic amino acid The characteristics of the group can indeed maintain a stable suspension state, and it is not easy to form precipitation and stratification, which can greatly reduce the proportion of insoluble proteins that are easy to precipitate, so there is no need for separation treatment. Since all proteins can exist in a suspension state, compared with In the past, the royal jelly solution was obtained by removing the sediment due to the large amount of layered sedimentation and obvious treatment. Obviously, the royal jelly solution of the present invention maintains the complete royal jelly component and has better nutritional value.

It should be added that when the pH value in the royal jelly solution is higher than the isoelectric point of protein, the pH value of the royal jelly solution can still be adjusted by using a food industry or medically permitted pH value regulator to make It is below the isoelectric point of the protein and keeps the solution in stable suspension.

<Concrete example one> the treatment result comparison of existing ferment and ferment of the present invention

Put the prepared royal jelly water suspension into 7 test tubes numbered in sequence A1, A2, A3, A4, A5, A6, A7 respectively, the royal jelly concentration of the royal jelly water suspension in each test tube All are 5.8wt%, the royal jelly water suspension in each test tube is processed as follows respectively:

A1: Add pepsin in an amount equivalent to 0.1wt% of the royal jelly aqueous suspension and act at a temperature of 45°C for 6 hours, then add acid protease in an amount equivalent to 0.1wt% of the royal jelly aqueous suspension (Using protease derived from Aspergillus Oryzae), after acting at a temperature of 45°C for 6 hours, the pH value of the solution was adjusted to 5.5, and heated at a temperature of 80°C for 10 minutes to terminate the enzyme action.

A2: The pH value of this royal jelly aqueous suspension is adjusted to 8, and the neutral protease (using the protease derived from Bacillus subtilis) (Prolease) and Trypsin, after acting at 45°C for 6 hours, adjusted the pH value of the solution to 5.5, and heated at 80°C for 10 minutes to terminate the enzyme action.

A3: No enzyme is added, the solution temperature is 55°C, the pH value is 4.0, and the state of the solution is observed after 3 hours.

A4: Add papaya enzyme in an amount equivalent to 0.15wt% of the royal jelly aqueous suspension, the solution temperature is 55°C, and the pH value is 4.1. After 0.5 hours, heat at 95°C for 5 minutes to terminate the enzyme action.

A5: The type of enzyme added, the amount added, the temperature of the solution, and the pH value are the same as A4, and the difference is that the enzyme action is terminated by heating after 1 hour of enzyme action.

A6: The type of enzyme added, the amount added, the solution temperature, and the pH value are the same as A4, and the difference is that after the enzyme acts for 3 hours, it is heated again to terminate the enzyme effect.

A7: The type of enzyme added, the amount added, the solution temperature, and the pH value are the same as A4, and the difference is that after the enzyme acts for 5 hours, it is heated again to terminate the enzyme effect.

Results: Referring to Figure 2, it shows the appearance of the solutions in test tubes A1 to A7 after the above treatments. Among them, the upper layer of A1 is clear, and the lower layer is obvious sediment. The upper layer of A2 is clear, while the lower layer is milky white opaque cloudy and contains sediment. A3 has a milky white precipitate at the bottom, and the upper layer is non-clear. Compared with A3, the amount of milky white sediment at the bottom of A4 has been reduced by more than half, and the upper layer is also non-clear. The entire test tubes of A5-A7 are non-clear without obvious layering, and there is no sediment at the bottom. Since A4～A7 are all treated with the papaya enzyme used in the present invention, the results shown in Figure 2 show that the royal jelly water suspension treated by the method of the present invention will be in a stable suspension state, so there will be no Obvious stratification, and the result of significantly reducing the amount of sediment at the bottom from A3 to A4, further shows that the aqueous suspension of royal jelly treated by the present invention can indeed act on insoluble proteins to improve the precipitation and stratification. Increasing the enzyme concentration or action time (as shown in A5-A7) can make the solution in the whole test tube appear free of precipitation and stratification, and present a curd-like and stable suspension state, while royal jelly treated with existing enzymes is suspended in water Liquid (such as A1 and A2), although the upper layer is clear, but there will still be a lot of sediment.

<Specific example 2> Test of solution stability, stability coefficient, active ingredient and suspension degree

Provide five royal jelly test solutions B1, B2, B3, B4 and B5 obtained by the manufacturing method of the present invention, and provide royal jelly test solutions C1, C2, C3, C4 and C5 obtained by general existing methods as Control comparison, and test and compare the precipitation rate, stability coefficient, decenoic acid content and stability, and suspension stability of the above test solutions. Wherein, the preparation methods of test solutions B1-B5 and test solutions C1-C5 are respectively as follows:

Test solution B1: Take 100g royal jelly and normal temperature water (about 25°C) and mix it uniformly to 1kg to make a 10wt% royal jelly aqueous suspension, and adjust its pH value to 4. After heating to 60°C, the This royal jelly aqueous suspension adds 10g of papaya enzyme (1% amount of the weight of royal jelly aqueous suspension) to obtain a royal jelly pre-reaction liquid, and implements enzyme action at 60°C for 3 hours, then makes the royal jelly The royal jelly solution obtained after the enzyme action was heated at 85° C. for 20 minutes to terminate the enzyme action therein.

Test solution B2: Mix 205g royal jelly with 45°C water and make it 1kg to make 20.5wt% royal jelly aqueous suspension, adjust its pH to 4 with citric acid or sodium citrate, then add pineapple Enzyme 10g (the amount that is 1% of the weight of royal jelly water suspension) forms royal jelly pre-reaction liquid, makes this pre-reaction liquid carry out ferment action at 55 ℃ of temperature for 1 hour, then this obtained after the action of pineapple enzyme The royal jelly solution was heated to 90°C and maintained at this temperature for 5 minutes to terminate the enzyme action.

Test solution B3: Mix 400g royal jelly with 1kg warm water (50°C) to prepare a royal jelly water suspension, and adjust its pH to 4. After heating to 50°C, add an amount equivalent to the royal jelly water suspension 5% bromelain by weight of the royal jelly aqueous suspension, and implement the enzyme action for 1 hour, then the solution obtained after the enzyme treatment was heated at 80° C. for 5 minutes to terminate the enzyme action.

Test solution B4: prepare 5.5wt% royal jelly aqueous suspension in the manner described in the preparation of test solution B3, after heating to 60°C, add 0.5% of the weight of the royal jelly aqueous suspension % papaya enzyme, and after implementing the enzyme action at 60°C for 50 minutes, then heated at a temperature of 90°C for 10 minutes to terminate the enzyme action.

Test solution B5: Take 58g royal jelly and prepare it as 5.8wt% royal jelly aqueous suspension, after heating to 55°C, add papaya enzyme equivalent to 0.15% of the weight of the royal jelly aqueous suspension to the royal jelly aqueous suspension , and implement the enzyme action at 55°C for 45 minutes, and then heat at 95°C for 5 minutes to terminate the enzyme action.

Test solution C1: prepare 10wt% royal jelly aqueous suspension in the manner described in test solution B1, and heat to a temperature of 60° C., perform hydrolysis for 3 hours without adding enzymes, and obtain milky white royal jelly solution, filter and dry Remove the precipitate and insoluble components.

Test solution C2: take royal jelly and prepare 20.5wt% royal jelly aqueous suspension with 45°C water, and add ethanol so that the amount of ethanol added is 35% (wt%) of the solution weight, and carry out at a temperature of 45°C Hydrolysis for 1 hour.

Test solution C3: Prepare 6 wt% royal jelly aqueous suspension, adjust its pH to 4, and let it stand in a water bath at a temperature of 50° C. for 1.5 hours.

Test solution C4: prepare 1kg of 10wt% royal jelly aqueous suspension, adjust its pH value to 4 with 20wt% sodium hydroxide solution, add 1g of pepsin to this solution, and implement enzyme treatment at 45°C6 After 1 hour, 1 g of acid protease (protease derived from Aspergillus Oryzae was used) was added, and enzyme treatment was carried out at a temperature of 45° C. for 6 hours. Finally, the pH value of the solution treated with the aforementioned enzyme was adjusted to 5.5 by using 20 wt% sodium hydroxide solution or 10 wt% citric acid solution, and heated at 80° C. for 10 minutes to inactivate the enzyme. Without filtering, a royal jelly solution in which the upper layer is clear and the lower layer is sediment can be obtained.

Test solution C5: Prepare 1 kg of royal jelly aqueous suspension of 10 wt%, and use 20 wt% sodium hydroxide solution to adjust its pH value to 8, then add neutral protease (using protease Prolease derived from Bacillus subtilis) to this solution and trypsin each 0.5g, after carrying out ferment treatment at 45 ℃ of temperature for 6 hours, to the solution after the aforementioned ferment treatment, its pH value is adjusted to 5.5, and heated at a temperature of 80°C for 10 minutes to inactivate the enzyme, without filtering, the upper layer is clear and the lower layer is milky white and opaque royal jelly solution.

(1) Precipitation rate test, stability evaluation and astringent sour taste evaluation

The precipitation rate of royal jelly solution indicates the long-term stability of the solution, usually expressed by its precipitation rate. In the royal jelly solution, the larger or heavier particles will settle at the bottom of the tube after being accelerated by gravity. Therefore, it can be used to judge the suspension stability of the solution. Generally speaking, the lower the precipitation rate of the solution, the better the stability of the solution. This test is used to measure the precipitation rate of the test solution B1-B5 prepared by the method of the present invention and the test solution C1-C5 prepared by the existing method, and thereby express the degree of emulsification and suspension of the solution. When performing the test, put the test solutions B1~B5 and test solutions C1~C5 in centrifuge tubes respectively, and centrifuge each test solution at a speed of 4000r.p.m. (E1) calculated. In addition, the stability evaluation and astringent sour taste test of the aforementioned test solution were carried out at the same time. The stability evaluation was to test the stability of the aforementioned test solution B1-B5 and test solution C1-C5 after being placed for 1 month. The degree of production is indicated. The astringent sour taste is the result of professional evaluation.

The test results are shown in Table-1 below.

Table-1 Sedimentation rate and beverage stability of test solution B1～B5 and C1～C5

Enzyme dosage Sedimentation rate Beverage stability Astringent sourness Test solution B1 ＜2% ◎ + Test solution B2 2.2% ○ + Test solution B3 ＜2% ◎ ++ Test solution B4 ＜2% ◎ + Test solution B5 ＜2% ◎ + Test solution C1 30% △ ++ Test Solution C2 10% △ +++ Test Solution C3 7% △ + Test solution C4 twenty three% △ ++ Test Solution C5 26% △ +

^* Evaluation of stability: ◎ means stable and very little precipitation; ○ means relatively unstable and a little precipitation;

△ means extremely unstable and precipitation occurs.

^** Astringent sourness: +++ means strong; ++ means medium; + means weak.

The results in Table-1 show that the royal jelly solution (test solution B1-B5) obtained after the action of papaya enzyme or bromelain enzyme, even after centrifugation, has a sedimentation rate comparable to that of the test solution not treated with the enzyme defined in the present invention. Compared with liquid C1-C5, it is still significantly lower. In addition, there is almost no sediment or agglutinate after the test solution B1-B5 is placed for 1 month, which shows that its stability is good. Compared with the test solution C1-C5, the royal jelly beverage product further made with the test solution B1-B5 Apparently less concern about precipitation or agglutination. In terms of flavor, the unique astringent sourness of the royal jelly solutions (test solutions B1-B5) after the enzyme treatment defined by the present invention is significantly lower than that of test solutions C1-C5, which shows that the preparation method of the present invention and the The royal jelly solution after the ferment treatment also has the effect of improving flavor. On the whole, the royal jelly solution obtained by the production method of the present invention has better stability and unique astringent sour taste after being made into a beverage than the royal jelly solution that has not been treated with the enzyme used in the method of the present invention.

(2) Stability factor

It is mainly used to indicate the solution stability of royal jelly solution in acidic environment. The higher the stability coefficient, the better the stability. This test is for the aforementioned test solutions B1~B5 and C1~C5 centrifuged at 3500r.p.m. ) to measure its absorbance at 660nm, and calculate it by the following formula:

The test results are shown in Figure 3. The results show that the royal jelly solution (test solution B1-B5) treated with the enzyme of the present invention has a higher stability than the royal jelly solution (test solution C1-C5) without the enzyme treatment of the present invention. better. Among them, the stability coefficient of test solution B4 is the best, followed by test solution B3. From the above results, it can be illustrated that the production method of the present invention and the enzyme used in the method have the effect of improving the solution stability of the royal jelly solution.

(3) Decenoic acid content and its stability

Decenoic acid is a biologically active substance in royal jelly, which has health care functions for the human body and is also an important indicator for evaluating the nutritional value of royal jelly-related products. This test is to test the content of decenoic acid and the stability of decenoic acid in the aforementioned test solutions B1-B5 and test solutions C1-C5 respectively. The content of decenoic acid is detected by HPLC (high performance liquid chromatography, brand: Agilent, model: 1200series) at a wavelength of 210nm with a C18 chromatography column (diameter: 4.6mm, length: 250mm), and then passed Obtained by comparison with the standard product inspection line.

Table-2 Decenoic acid content and stability test results of test solutions B1~B5 and C1~C5

Group Decenoic acid content (mg/g) Decenoic Acid Stability   Original Royal Jelly 6.2 Test Solution B1 6.2 +++   Test solution B2 6.0 ++   Test Solution B3 6.2 +++   Test Solution B4 6.2 +++   Test Solution B5 6.2 +++

Test solution C1 4.9 + Test Solution C2 4.8 + Test solution C3 5.5 + Test Solution C4 5.9 ++ Test Solution C5 6.0 ++

^* Stability assessment: expressed according to the degree of decline. Stability: +++ indicates that the decenoic acid content remains unchanged after one month; ++ indicates that the decenoic acid content decreases by about <1% after one month; + indicates that the decenoic acid content decreases by about >1% after one month.

The results in Table-2 show that the royal jelly solution (test solutions B1-B5) prepared by the production method of the present invention has no significant difference in the decenoic acid content compared with the decenoic acid content of the original royal jelly after being stored at room temperature for one month , showing that the royal jelly solution treated with the enzyme in the production method of the present invention not only can retain the active ingredient of royal jelly-decenoic acid, but also has better content stability than the royal jelly solution that is not treated by the method of the present invention. However, the content of decenoic acid in the royal jelly solutions of test solutions C1-C5 has a slight downward trend, and after storage at normal temperature, the content also has a tendency to decline. This shows that the decenoic acid content in the royal jelly solution obtained by the production method of the present invention is relatively stable and has better nutritional quality.

(4) Suspension degree test

For the aforementioned test solutions B1-B5 and test solutions C1-C5, respectively test the suspension degree of their solutions. The degree of suspension is measured with a Turbidity meter (model: HACH 2100Q), which is mainly measured by the principle of light scattering, and the degree of light scattering when it enters the water body is used as the standard for judging the degree of suspension. In the suspension state, the solution will hinder the penetration of light, so that the turbidity of the solution will be high, and the unit is the standard turbidity unit (Nephelometric Turbidity Unit, NTU). Wherein, the royal jelly solutions of test solution C4 and test solution C5 are firstly filtered to remove sediment, and then the turbidity is measured for the clear and transparent royal jelly solution without sediment.

Table-3 Turbidity and Suspension Stability of Test Solution B1～B5 and C1～C5

Group Turbidity (NTU) Stability Test Solution B1 755.0 +++ Test Solution B2 752.1 ++ Test Solution B3 755.3 +++ Test Solution B4 755.1 +++ Test solution B5 753.0 +++ Test Solution C1 116 + Test solution C2 85 +

Test Solution C3 109 + Test solution C4 29.3 + Test solution C5 30.1 +

^* Stability assessment: expressed according to the degree of decline. Stability: +++ indicates that the turbidity measured after one month remains unchanged; ++ indicates that the turbidity change range is about <1% when measured after one month; + indicates that the turbidity change range is about >1% when measured after one month %.

The results in Table-3 show that the royal jelly solutions (test solutions B1-B5) prepared by the production method of the present invention have no significant change in turbidity after being stored at room temperature for one month. Test solutions B1-B5 can maintain a stable suspension state for a long time, and are not easy to form stratification or precipitation. However, although the royal jelly solutions of test solutions C1-C5 had lower original turbidity (that is, the solution was relatively clear) due to precipitation and stratification, after one month of storage at room temperature, the measured turbidity There is still an upward trend, indicating that after the solution is stored, there is still precipitation in the clarified liquid and there is a phenomenon of white turbidity, which leads to an increase in turbidity. Furthermore, it can be reasonably inferred that the components in the test solutions C1-C5 cannot exist stably. According to this, it is shown that the protein molecules in the royal jelly solution obtained by the production method of the present invention do have the effect of maintaining a stable suspension. Obviously, the components in the solution can exist stably for a long time, so there is no need to filter or centrifuge to remove, and this can be used The characteristics of a stable suspension and stable ingredients are used to produce royal jelly-related drinks or liquid products in a way that retains the complete royal jelly protein.

<Specific example 3> Identification of protein

It mainly adopts Peptide Mass Fingerprinting technology, and uses matrix-assisted laser desorption free time-of-flight mass spectrometer (MALFI-TOF-MASS, brand: Applied Biosystems Voyager-DE PR, operating mode: linear, polar : positive charge, voltage: 2000V, mass range: 500-15000Da) to identify the protein quality in the prepared test solution. The protein identity identification test was carried out for the royal jelly water suspension without enzyme action-test solution D1, and the royal jelly solution obtained through different types and concentrations of enzymes under different action conditions-test solutions D2-D7. Test solutions D1-D7 are as follows:

Test solution D1: 6wt% royal jelly suspension without enzyme action.

Test solution D2: prepare 6wt% royal jelly aqueous suspension, add papaya enzyme equivalent to 0.1% of the weight of royal jelly aqueous suspension, and carry out enzyme action at a temperature of 50-55° C. for 30 minutes.

Test solution D3: prepare 6wt% royal jelly aqueous suspension, add papaya enzyme equivalent to 0.1% of the weight of royal jelly aqueous suspension, and carry out enzyme action at a temperature of 50-55° C. for 3 hours.

Test solution D4: Prepare 6wt% royal jelly aqueous suspension, add papaya enzyme equivalent to 1% of the weight of royal jelly aqueous suspension, and carry out enzyme action at a temperature of 50-55° C. for 30 minutes.

Test solution D5: Prepare 6wt% royal jelly aqueous suspension, add papaya enzyme equivalent to 1% of the weight of royal jelly aqueous suspension, and carry out enzyme action at a temperature of 50-55° C. for 3 hours.

Test solution D6: prepare 6wt% royal jelly aqueous suspension, and add 0.1% bromelain to it, which is equivalent to the weight of royal jelly aqueous suspension, and carry out enzyme action at a temperature of 50-55° C. for 1 hour.

Test solution D7: prepare 6wt% royal jelly aqueous suspension, and add 1% bromelain to it, which is equivalent to the weight of royal jelly aqueous suspension, and carry out enzyme action at a temperature of 50-55° C. for 1 hour.

The above test solutions D1-D7 were analyzed by mass spectrometry after the desalting procedures of MALDI-TOF MS (brand name: Applied Biosystems Voyager-DE PR) using the existing protein identity identification method, that is, peptide mass fingerprinting technology (Peptide Mass Fingerprinting). and find out its characteristic protein mass

Comparing the mass spectrograms in Figure 4 and Figures 5 to 10, it can be seen that the royal jelly solution treated with enzymes and the royal jelly aqueous suspension without enzyme treatment have a protein mass between 1000 and 1900 m/z (mass-to-charge ratio )(mass tocharge ratio), 2000～2900m/z, 3100～3600m/z, 4200～4500m/z, 6000～6400m/z have significant differences, and it is judged that the peak signal here is after the action of enzyme The quality of the cut protein molecules, after processing by the peptide mass fingerprint technology for protein identity identification, is evaluated as 1016m/z±0.5%, 1214m/z±0.5%, 2016m/z±0.5%, 2032m/z±0.5% %, 3477m/z±0.5%, 6030±0.5%m/z, 6314m/z±0.5%, 7413±0.5%, 8640m/z±0.5% are the characteristic proteins of the royal jelly solution produced by the production method of the present invention quality. That is, the royal jelly solution obtained through the enzyme treatment in the production method of the present invention contains protein molecules of the above-mentioned characteristic protein quality.

To sum up the above, the royal jelly solution of the present invention and its manufacturing method can obtain the following effects and advantages, thereby achieving the purpose of the present invention:

One, as can be clearly seen from the processing method of the above-mentioned embodiment, the present invention does not need to use two Only one or more kinds of enzymes can achieve the predetermined treatment effect. In fact, the present invention only needs to add one kind of enzyme to process the raw royal jelly into a royal jelly solution with stable suspension and stable components. The quality of the prepared royal jelly solution is quite stable, so it is suitable for retaining the whole queen bee It is further made into royal jelly related products by means of milk components. Because only one kind of enzyme can be used for processing, and no additional filtration or centrifugation procedures are required, the present invention can reduce the cost of raw materials, simplify the process steps, and the quality of the obtained product is stable. Therefore, compared with the prior art, the present invention It does have better economic and environmental benefits in practical application.

2. The present invention treats the royal jelly aqueous suspension with an enzyme containing endocysteine, and only needs to add a single enzyme to the royal jelly aqueous suspension to obtain non-transparent, water-setting The milky full royal jelly solution can simplify the process and shorten the high cost caused by the long-term manufacturing process, not only can improve the process efficiency but also save the manufacturing cost, which is more in line with the principle of commercial economic benefits, and has better economic benefits.

3. The royal jelly solution obtained by the production method of the present invention can not only be made into a product by retaining the complete components, but also the results of the above test and analysis show that the royal jelly solution treated with the enzyme of the present invention has the same stability as the solution itself. Stability coefficient, content of active ingredients and stability of active ingredients, as well as long-term maintenance and stability of the suspended state, all have very stable performance, showing that the royal jelly solution of the present invention not only has more complete nutritional ingredients, but also has a relatively stable product quality characteristics.

4. The royal jelly solution prepared by the present invention can maintain a stable suspension state, no need to remove sediment and filter, and can completely retain the royal jelly components, effectively avoiding the waste of precious ingredients and the recovery rate. Compared with In the past, two or more enzymes were used to act on the royal jelly aqueous suspension to obtain a clarified royal jelly drink. The present invention not only has a simpler and more economical process, but also has higher nutritional value, and the manufacturing cost of this method Low and suitable for mass production, it is of great practical value in the manufacturing and production of the food industry.

It should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. the manufacture method of queen bee breast solution; It is characterized in that, this manufacture method comprises the following step:

One, a kind of queen bee dairy milk starting material is mixed to obtain a kind of queen bee whey suspension with water;

Two, interpolation contains the ferment of NAC restriction endonuclease to obtain a kind of queen bee breast pre-reaction liquid in this queen bee whey suspension; And

Three, this queen bee breast pre-reaction liquid is reacted under 20 ℃～70 ℃ of temperature, to obtain a kind of curdy and to be the queen bee breast solution of stable suspersion state.

2. the manufacture method of queen bee as claimed in claim 1 breast solution, it is characterized in that: used ferment is Fructus Chaenomelis ferment, Bromelain, fig ferment in step 2, or the combination of aforementioned ferment.

3. the manufacture method of queen bee as claimed in claim 2 breast solution is characterized in that: the addition of the ferment in step 2 is 0.01%～90% of the weight of this queen bee whey suspension.

4. the manufacture method of queen bee as claimed in claim 1 breast solution is characterized in that: be first the pH value of this queen bee whey suspension to be adjusted into 2～8 in step 2, add ferment again.

5. the manufacture method of queen bee as claimed in claim 1 breast solution is characterized in that: the concentration of the queen bee dairy milk starting material of the queen bee whey suspension of preparing in step 1 is 0.1～40wt%.

6. the manufacture method of queen bee as claimed in claim 1 breast solution, it is characterized in that: in step 3, after making this queen bee breast pre-reaction liquid under 20 ℃～70 ℃ of temperature, react 0.5～10 hour, suppress ferment continuation effect in a kind of mode that is selected from the following group: this queen bee breast pre-reaction liquid is heated to temperature 70 C～110 ℃, and the pH in this queen bee breast pre-reaction liquid is adjusted to less than 2 or greater than 8.

7. queen bee breast solution is to add acquisition after the ferment reaction in a kind of queen bee whey suspension that a kind of queen bee dairy milk starting material and water mix; It is characterized in that:

The ferment that adds in this queen bee whey suspension is the ferment that contains the NAC restriction endonuclease, and this queen bee breast solution is curdy and is the stable suspersion state, and comprises the protein with following characteristic protein matter quality: 1214m/z ± 0.5% and/or 2032m/z ± 0.5%.

8. queen bee as claimed in claim 7 breast solution is characterized in that: this queen bee breast solution also comprises and has following at least wherein a kind of protein of characteristic protein matter quality: 1016m/z ± 0.5%, 2016m/z ± 0.5%, 3477m/z ± 0.5%, 6030m/z ± 0.5%, 6314m/z ± 0.5%, 7413m/z ± 0.5% and 8640m/z ± 0.5%.

9. queen bee as claimed in claim 7 breast solution, it is characterized in that: the pH value of this queen bee breast solution is 3～5.

10. queen bee as claimed in claim 7 breast solution, it is characterized in that: the addition that contains the ferment of NAC restriction endonuclease is 0.01%～90% of the weight of this queen bee whey suspension, and the ferment that contains the NAC restriction endonuclease that adds is Fructus Chaenomelis ferment, Bromelain, fig ferment, or the combination of aforementioned ferment.

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