阅读说明：本技术 一种液体乳制品的生产方法 (Production method of liquid dairy product ) 是由 金磊 巴根纳 于 2019-11-29 设计创作，主要内容包括：本发明提供了一种液体乳制品的生产方法,尤其涉及一种富含乳铁蛋白的液体乳制品的生产方法。目前现有技术均存在：(1)原本存在于乳制品中的天然的乳铁蛋白在除菌过程中失活,需外源添加,既浪费又无法满足人们对自然食品的需求；(2)现有技术中乳制品除菌需两次较长时间杀菌,导致乳制品产生不愉快的蒸煮味,进而失去鲜牛乳原有的新鲜口感。为解决上述问题,本发明提供了一种方法,获得富含乳铁蛋白的液体乳制品。本发明方法满足了人们对生活和健康的日益重视,对自然的(不是外加的而是本身含有的)、健康的乳制品的需求。(The invention provides a production method of a liquid dairy product, and particularly relates to a production method of a liquid dairy product rich in lactoferrin. The prior art at present all has: (1) the natural lactoferrin originally existing in the dairy product is inactivated in the degerming process, and needs to be added externally, so that the natural lactoferrin is wasted and cannot meet the requirements of people on natural foods; (2) in the prior art, the dairy product needs to be sterilized twice for a long time, so that the dairy product generates unpleasant cooking taste, and the original fresh taste of fresh milk is lost. In order to solve the problems, the invention provides a method for obtaining a liquid dairy product rich in lactoferrin. The method of the invention meets the increasing importance of people on life and health, and the demand of natural (not added but contained) and healthy dairy products.)

1. A preparation method of a liquid dairy product comprises the following steps:

(1) extracting lactoferrin from the raw milk to obtain lactoferrin and milk from which the lactoferrin is extracted respectively;

(2) performing aseptic treatment on the extracted lactoferrin;

(3) sterilizing the raw milk after extracting the lactoferrin at the temperature of 155-165 ℃ for 0.07-0.14 s;

(4) and (3) adding the lactoferrin subjected to the aseptic treatment in the step (2) into the raw milk subjected to the sterilization treatment in the step (3) and subjected to lactoferrin extraction to obtain the liquid dairy product rich in lactoferrin.

2. The method of claim 1, wherein the step (1) comprises the steps of:

(a) cleaning milk: carrying out centrifugal milk cleaning treatment on the raw milk;

(b) separation: centrifuging the raw milk of step (a) to separate milk fat from skim milk;

(c) filtering the skim milk: filtering a small amount of fat particles remained in the skim milk by using a filter bag;

(d) extraction of lactoferrin: extracting the lactoferrin component from the skim milk of step (b) to obtain active lactoferrin.

3. The method according to claim 1, wherein the sterilization treatment of step (2) is membrane filtration sterilization.

4. The method as claimed in claim 1, wherein the step (3) comprises the steps of:

(a) mixing: mixing the skim milk with lactoferrin with milk fat;

(b) preheating: heating the mixture of step (a) to 55-60 ℃;

(c) homogenizing: homogenizing the mixture of step (b) at 150-;

(d) and (3) sterilization: sterilizing the homogenized milk at the temperature of 160 ℃/0.07-0.14s at 155-;

(e) cooling the sterilized feed liquid to 20-25 deg.C, and storing in sterile tank.

5. The method of claim 1, wherein the step (4) comprises the steps of:

(a) mixing: mixing the sterilized liquid lactoferrin and the sterilized milk on line or in a sterile tank;

(b) canning: and (b) carrying out aseptic filling on the mixed solution in the step (a).

6. A method according to claim 2, wherein the filtration of skim milk is performed by filtering off small amounts of fat particles remaining in skim milk using a filter bag of 1-5 μm, preferably 1 μm.

7. A process according to claim 1 or 2 wherein the extraction of lactoferrin is carried out by a process selected from: salting out, organic solvent, ultrafiltration, ion exchange resin chromatography, affinity chromatography, immobilized monoclonal antibody, and mixed mode expanded bed adsorption.

8. The method according to claim 3, wherein the membrane filtration sterilization is performed by using a sterilization membrane of 0.1-0.45 μm.

Technical Field

The invention relates to a production method of a liquid dairy product, in particular to a production method of a liquid dairy product containing active lactoferrin and having a mouthfeel similar to that of a pasteurized dairy product.

Background

Lactoferrin (LF) is an important non-heme iron-binding glycoprotein in milk, and is a monomeric glycoprotein with bactericidal activity in neutrophil granules. It has a molecular weight of 80kDa and is mainly expressed and secreted by mammary epithelial cells. In 1939 Sorensen et al isolated whey protein and Polis et al isolated Lp also partially purified red protein, but until 1959 Groves chromatographed pure red material, which was identified as an iron-binding glycoprotein called lactoferrin. Lactoferrin not only participates in the transport of iron, but also has strong biological functions of broad-spectrum antibiosis, antioxidation, anticancer, immune system regulation and the like, and is considered as a novel antibacterial and anticancer drug and a food additive with great development potential. Lactoferrin has poor thermal stability, and its degree of thermal denaturation gradually increases with increasing temperature in the range of 65 ℃ to 95 ℃.

Lactoferrin is widely distributed in mammalian milk (raw milk content: 0.0128-0.0159%), but scientific studies have confirmed that raw milk is a high risk food because it does not kill harmful bacteria. Infants, elderly and infirm people, pregnant women and people with immune system problems may develop the condition if they drink raw milk. Therefore, raw milk cannot be directly eaten, and heating sterilization treatment is required before raw milk is eaten. However, the heating process has a problem that the lactoferrin in the milk loses the original activity after being heated in the process of heating and sterilizing the raw milk, and the inactivated lactoferrin has no effect and action on human bodies.

Although the prior art production processing method can produce and process the lactoferrin-rich liquid dairy product. However, the production methods of the prior art are all as follows: the liquid milk material liquid is sterilized first, and then the sterile lactoferrin is added into the sterilized liquid milk material liquid in a sterile online adding mode, for example, the production methods of patent numbers 200810094034.6 and 201110083557.2, although the liquid milk product containing the lactoferrin can be finally obtained by the methods.

However, the prior preparation methods all have the following problems:

firstly, sterilizing milk originally containing active lactoferrin to inactivate the original active lactoferrin in the milk, and then adding the active lactoferrin into the sterilized (lactoferrin inactivated) milk in a mode of adding lactoferrin. This production method is a great waste.

Secondly, the temperatures of pasteurization and high-temperature sterilization are both 121-150 ℃, and the holding time is 4-15 s. And the pasteurization process of keeping the temperature at 72-90 ℃ for 5-20s exists before high-temperature sterilization. The sterilization process for twice sterilizing the milk and keeping the sterilization for a long time can cause the liquid dairy product to generate unpleasant cooking flavor, thereby losing the original fresh taste of the fresh milk.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for preparing a liquid dairy product containing active lactoferrin, by which the liquid dairy product containing active lactoferrin can be produced. The liquid dairy product produced by the method not only contains the active lactoferrin, but also has fresh mouthfeel compared with the traditional production method in the aspect of product flavor and mouthfeel. Compared with the traditional liquid dairy product containing active lactoferrin in the production method, the invention can effectively utilize the active lactoferrin component contained in the milk before sterilization. Instead of the traditional process, the milk containing the active lactoferrin is sterilized firstly, so that the lactoferrin activity in the milk is inactivated, and then the aseptic lactoferrin is added externally. In addition, the traditional production method has more times of sterilizing the milk before adding the lactoferrin. The sterilization temperature and the sterilization temperature holding time are long. The milk is sterilized for more than one time and the sterilization and retention time is long, so that the traditional liquid dairy product generates unpleasant cooking flavor.

The invention provides a preparation method of a liquid dairy product, which comprises the following steps:

(1) extracting lactoferrin from the raw milk to obtain lactoferrin and milk from which the lactoferrin is extracted respectively;

(2) performing aseptic treatment on the extracted lactoferrin;

(3) sterilizing the raw milk after extracting the lactoferrin at the temperature of 155-165 ℃ for 0.07-0.14 s;

(4) and (3) adding the lactoferrin subjected to the aseptic treatment in the step (2) into the raw milk subjected to the sterilization treatment in the step (3) and subjected to lactoferrin extraction to obtain the liquid dairy product rich in lactoferrin.

In one embodiment, wherein the step (1) comprises the steps of:

(a) cleaning milk: carrying out centrifugal milk cleaning treatment on the raw milk;

(b) separation: centrifuging the raw milk of step (a) to separate milk fat from skim milk;

(c) filtering the skim milk: filtering a small amount of fat particles remained in the skim milk by using a filter bag;

(d) extraction of lactoferrin: extracting the lactoferrin component from the skim milk of step (b) to obtain active lactoferrin.

In a preferred embodiment, wherein the extraction of lactoferrin is performed by a method selected from the group consisting of: salting out, organic solvent, ultrafiltration, ion exchange resin chromatography, affinity chromatography, immobilized monoclonal antibody, and mixed mode expanded bed adsorption.

In a preferred embodiment, wherein the filtering of skim milk is to filter off a small amount of fat particles remaining in skim milk using a filter bag of 1-5 μm, preferably 1 μm.

In one embodiment, wherein the sterilization treatment of step (2) is membrane filtration sterilization.

In a preferred embodiment, the membrane filtration sterilization is implemented by using a sterilization membrane with the diameter of 0.1-0.45 μm.

In one embodiment, wherein the step (3) comprises the steps of:

(a) mixing: mixing the skim milk with lactoferrin with milk fat;

(b) preheating: heating the mixture of step (a) to 55-60 ℃;

(c) homogenizing: homogenizing the mixture of step (b) at 150-;

(d) and (3) sterilization: sterilizing the homogenized milk at the temperature of 160 ℃/0.07-0.14s at 155-;

(e) cooling the sterilized feed liquid to 20-25 deg.C, and storing in sterile tank.

In one embodiment, wherein the step (4) comprises the steps of:

(a) mixing: mixing the sterilized liquid lactoferrin and the sterilized milk on line or in a sterile tank;

(b) canning: and (b) carrying out aseptic filling on the mixed solution in the step (a).

In one embodiment, wherein the extraction of lactoferrin is performed by a method selected from the group consisting of: salting out, organic solvent, ultrafiltration, ion exchange resin chromatography, affinity chromatography, immobilized monoclonal antibody, and mixed mode expanded bed adsorption.

In a preferred embodiment, the invention provides a method for manufacturing a liquid dairy product, in particular to a method for producing a liquid dairy product containing active lactoferrin. The method specifically comprises the following steps:

(1) cleaning milk: and (4) centrifuging and purifying the raw milk by a milk purifier.

(2) Separation: and (3) carrying out centrifugal separation on the raw milk to separate the milk fat from the skim milk.

(3) And (3) lactoferrin extraction: the lactoferrin component is extracted from skim milk to obtain the active lactoferrin. For example: salting out, organic solvent, ultrafiltration, ion exchange resin chromatography, affinity chromatography, immobilized monoclonal antibody, and mixed mode expanded bed adsorption.

(4) And (3) lactoferrin sterilization: and (3) performing membrane filtration sterilization on the liquid lactoferrin.

(5) Mixing: the skim milk after the lactoferrin extraction is mixed with milk fat on line.

(6) Preheating: heating the mixed raw milk to 55-60 ℃.

(7) Homogenizing: the milk without lactoferrin after mixing and heating to 55-60 ℃ is homogenized at 150-180 bar.

(8) And (3) sterilization: sterilizing homogenized milk at 160 deg.C/0.07-0.14 s at 155-.

(9) Sterile mixed lactoferrin: mixing the sterilized liquid lactoferrin and the sterilized milk on line or in a sterile tank.

(10) Filling: and (4) aseptically filling the mixed feed liquid to obtain the liquid dairy product which does not need to add lactoferrin from an external source and has fresh mouthfeel.

In actual production, persons skilled in the art can understand that some relevant process steps can be adjusted or increased or decreased on the premise that qualified products can be obtained.

Advantageous effects of the invention

1. The invention solves the problems that the prior production method of the liquid dairy product containing the active lactoferrin component comprises the following steps: the milk is firstly sterilized to inactivate the active lactoferrin in the milk, and then the wasteful and uneconomical production and processing method of the active lactoferrin is carried out.

2. The method not only meets the increasing importance of people on life and health, but also meets the requirements on natural (not added but contained) healthy dairy products. Meanwhile, the production and processing technology in the field is perfected.

3. The liquid dairy product manufactured by the method has a clean label, and the invention can realize the purpose of adding little or no food additives in the product as far as possible, thereby maintaining the natural attribute of the food in the label ingredient column.

4. The invention solves the problems that the prior production method of the liquid dairy product containing the active lactoferrin component comprises the following steps: the milk before the lactoferrin is added is sterilized twice, and the holding time of each sterilization is longer. So that the produced liquid dairy product generates unpleasant cooking flavor.

Drawings

Fig. 1 shows that the sterile lactoferrin solution with activity is added back to the aseptic tank containing the sterile milk, and then mixed with the sterilized milk in the aseptic tank for aseptic filling.

Fig. 2 shows the sterile solution of active lactoferrin added back to the sterile feed line and filled aseptically.

FIG. 3 shows a liquid dairy product produced without lactoferrin extraction from milk prior to ultra-high temperature sterilization in the conventional production process 1.

Fig. 4 is a production process of a traditional production process 2, which is to perform ultra-high temperature sterilization treatment on milk and then additionally add lactoferrin.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

The embodiment provides a liquid dairy product, which is prepared by the following steps:

1. separating the raw milk to obtain skim milk and cream respectively.

2. And (3) filtering the skim milk by using a filter bag with the diameter of 1 mu m to obtain filtered skim milk.

3. The method comprises the following steps of carrying out adsorption, elution, concentration, desalination and the like on skim milk to obtain active lactoferrin liquid from the skim milk, and specifically comprises the following steps:

3.1 ion exchange

Heating the skim milk obtained in the step 2 to 4 ℃; carrying out ion exchange treatment on skim milk at the temperature of 4 ℃ by using strong cation exchange resin so as to enable lactoferrin to be adsorbed on an ion exchange column; the milk retention time in the ion exchange column was 6 minutes.

3.2 gradient elution

Performing gradient elution on the ion exchange column to obtain a salt solution containing lactoferrin, which comprises the following steps:

a. pre-eluting with water;

b. performing a first elution with a 30 mass% potassium chloride solution;

c. the second elution was performed with a 80 mass% potassium chloride solution.

3.3 desalination treatment

Desalting the obtained lactoferrin-containing salt solution by using an organic membrane to obtain lactoferrin, wherein the membrane concentration multiple is 10. 4. The liquid lactoferrin was sterilized by filtration using a sterilizing filter having a pore size of 0.22 μm to obtain a sterilized liquid lactoferrin. Then, the sterile liquid lactoferrin is pumped into a sterile tank for sterile temporary storage.

5. Mixing the skim milk with lactoferrin and dilute cream on line.

6. Mixing skim milk and cream, and preheating at 60 deg.C.

7. Homogenizing the preheated milk, wherein the homogenizing temperature and the homogenizing pressure are as follows: 60 ℃ at 250 bar.

8. Sterilizing homogenized milk at 159 deg.C/0.09 s.

9. And cooling the sterilized milk at 25 ℃.

10. And (4) carrying out sterile temporary storage on the sterilized and cooled cow milk.

11. Sterile liquid lactoferrin is pumped into a sterile tank for storage of sterile milk for sterile mixing.

12. After mixing, the milk containing the active lactoferrin is aseptically filled. And (4) carrying out aseptic filling to obtain the liquid dairy product. The specific process is shown in figure 1.

Example 2

The embodiment provides a liquid dairy product, which is prepared by the following steps:

1. separating the raw milk to obtain skim milk and cream respectively.

2. And (3) filtering the skim milk by using a filter bag with the diameter of 1 mu m to obtain filtered skim milk.

3. The method comprises the following steps of carrying out adsorption, elution, concentration, desalination and the like on skim milk to obtain active lactoferrin liquid from the skim milk, and specifically comprises the following steps:

3.1 ion exchange

Heating the skim milk obtained in the step 2 to 4 ℃; carrying out ion exchange treatment on skim milk at the temperature of 4 ℃ by using strong cation exchange resin so as to enable lactoferrin to be adsorbed on an ion exchange column; the milk retention time in the ion exchange column was 7 minutes.

3.2 gradient elution

Performing gradient elution on the ion exchange column to obtain a salt solution containing lactoferrin, which comprises the following steps:

a. pre-eluting with water;

b. performing a first elution with a 30 mass% potassium chloride solution;

c. the second elution was performed with a 80 mass% potassium chloride solution.

3.3 desalination treatment

Desalting the obtained lactoferrin-containing salt solution by using an organic membrane to obtain lactoferrin, wherein the membrane concentration multiple is 11. 4. The liquid lactoferrin was sterilized by filtration using a sterilizing filter having a pore size of 0.22 μm to obtain a sterilized liquid lactoferrin. Then, the sterile liquid lactoferrin is pumped into a sterile tank for sterile temporary storage.

5. Mixing the skim milk with lactoferrin and dilute cream on line.

6. The skim milk and the cream are mixed and then preheated, wherein the preheating temperature is 57 ℃.

7. Homogenizing the preheated milk, wherein the homogenizing temperature and the homogenizing pressure are as follows: 57 ℃ C/280 bar.

8. And (3) sterilizing the homogenized milk at the temperature of 158 ℃/0.1 s.

9. Cooling the sterilized milk at 30 ℃.

10. And (4) carrying out sterile temporary storage on the sterilized and cooled cow milk.

11. Sterile liquid lactoferrin is aseptically added into a material pipeline before filling, so that the sterile lactoferrin and the sterilized milk are mixed on line.

12. And (3) carrying out sterile filling on the mixed sterile milk containing the active lactoferrin. And (4) carrying out aseptic filling to obtain the liquid dairy product. The specific process is shown in FIG. 2.

The traditional production process 1: the liquid dairy product produced without lactoferrin extraction of cow milk before ultra-high temperature sterilization treatment is shown in figure 3.

The traditional production process 2: the production process comprises the steps of carrying out ultra-high temperature sterilization treatment on milk and then additionally adding lactoferrin. The specific process is shown in FIG. 4.

Example 3

The embodiment provides a liquid dairy product, which is prepared by the following steps:

1. separating the raw milk to obtain skim milk and cream respectively.

2. And (3) filtering the skim milk by using a filter bag with the diameter of 1 mu m to obtain filtered skim milk.

3. The skim milk is subjected to adsorption, elution, concentration, desalination and other steps, and active lactoferrin liquid is obtained from the skim.

3.1 ion exchange

Heating the skim milk obtained in the step 2 to 4 ℃; carrying out ion exchange treatment on skim milk at the temperature of 4 ℃ by using strong cation exchange resin so as to enable lactoferrin to be adsorbed on an ion exchange column; the milk retention time in the ion exchange column was 6.5 minutes.

3.2 gradient elution

Performing gradient elution on the ion exchange column to obtain a salt solution containing lactoferrin, which comprises the following steps:

a. pre-eluting with water;

b. performing a first elution with a 30 mass% potassium chloride solution;

c. the second elution was performed with a 80 mass% potassium chloride solution.

3.3 desalination treatment

Desalting the obtained lactoferrin-containing salt solution by using an organic membrane to obtain lactoferrin, wherein the membrane concentration multiple is 11.

4. The liquid lactoferrin was sterilized by filtration using a sterilizing filter having a pore size of 0.22 μm to obtain a sterilized liquid lactoferrin. Then, the sterile liquid lactoferrin is pumped into a sterile tank for sterile temporary storage.

5. The skim milk from which lactoferrin was extracted was then mixed with cream on-line.

6. Mixing skim milk and cream, and preheating at 58 deg.C.

7. Homogenizing the preheated milk, wherein the homogenizing temperature and the homogenizing pressure are as follows: 58 ℃ C/300 bar.

8. Sterilizing homogenized cow milk at 155 deg.C/0.14 s.

9. And cooling the sterilized milk at 25 ℃.

10. And (4) carrying out sterile temporary storage on the sterilized and cooled cow milk.

11. Sterile liquid lactoferrin is pumped into a sterile tank for storage of sterile milk for sterile mixing.

12. After mixing, the milk containing the active lactoferrin is aseptically filled.

Example 4

The embodiment provides a normal-temperature liquid dairy product, which is prepared by the following steps:

1. separating the raw milk to obtain skim milk and cream respectively.

2. And (3) filtering the skim milk by using a filter bag with the diameter of 1 mu m to obtain filtered skim milk.

3. The skim milk is subjected to adsorption, elution, concentration, desalination and other steps, and active lactoferrin liquid is obtained from the skim. 3.1 ion exchange

Heating the skim milk obtained in the step 2 to 4 ℃; carrying out ion exchange treatment on skim milk at the temperature of 4 ℃ by using strong cation exchange resin so as to enable lactoferrin to be adsorbed on an ion exchange column; the milk retention time in the ion exchange column was 6.5 minutes.

3.2 gradient elution

Performing gradient elution on the ion exchange column to obtain a salt solution containing lactoferrin, which comprises the following steps:

a. pre-eluting with water;

b. performing a first elution with a 30 mass% potassium chloride solution;

c. the second elution was performed with a 80 mass% potassium chloride solution.

3.3 desalination treatment

Desalting the obtained lactoferrin-containing salt solution by using an organic membrane to obtain lactoferrin, wherein the membrane concentration multiple is 11. 4. The liquid lactoferrin was sterilized by filtration using a sterilizing filter having a pore size of 0.22 μm to obtain a sterilized liquid lactoferrin. Then, the sterile liquid lactoferrin is pumped into a sterile tank for sterile temporary storage.

5. Mixing the skim milk with lactoferrin and dilute cream on line.

6. The skim milk and the cream are mixed and then preheated, wherein the preheating temperature is 57 ℃.

7. Homogenizing the preheated milk, wherein the homogenizing temperature and the homogenizing pressure are as follows: 57 ℃ C/280 bar.

8. And (3) sterilizing the homogenized milk at the temperature of 160 ℃/0.07 s.

9. Cooling the sterilized milk at 30 ℃.

10. And (4) carrying out sterile temporary storage on the sterilized and cooled cow milk.

11. Sterile liquid lactoferrin is aseptically added into a material pipeline before filling, so that the sterile lactoferrin and the sterilized milk are mixed on line.

12. And (3) carrying out sterile filling on the mixed sterile milk containing the active lactoferrin.

EXAMPLE 5 comparison of Lactoferrin content

Compared with the data of the lactoferrin content and the hygiene index in the liquid dairy product prepared by the traditional production process 1, the production method of the invention has the advantages that the specific comparison data of the lactoferrin content is shown in the table 1, and the specific comparison data of the hygiene index is shown in the table 2.

The production method a: the production method of the milk comprises the steps of extracting lactoferrin from milk before sterilization and then adding the lactoferrin (namely the method in the embodiment 1).

The production method b: the production method of extracting lactoferrin from cow milk before sterilization (traditional production process 1).

As shown in the results in tables 1 and 2 below, in the conventional production process 1 in which the activity of the original lactoferrin was not ensured or no lactoferrin was added externally, the lactoferrin content in the milk was extremely low after the ultra-high temperature sterilization treatment. In sharp contrast to the production method of the present invention. Compared with the traditional sterilization method, the sterilization method has no difference in sterilization effect.

TABLE 1 comparison data of lactoferrin content in two production processes

TABLE 2 comparison of microorganisms (Total number of colonies) data for both production methods

Statistical methods for colony counts were performed using methods conventional in the art: number of colonies in the plate to total number of colonies. 1. 2, 3 if 1 is not long, the result is recorded as < 1.

Example 4 mouthfeel evaluation

The examples 1 and 2 and the conventional process 2 are taken as taste test samples, and the number of test persons is 200, and the products are evaluated respectively from the nutrition feeling, the color and luster of the products, the flavor of the products, the mouthfeel of fresh milk and the preference degree of the products in an anonymous mode.

The specific evaluation is shown in Table 3, wherein,

the nutrition feeling, the product color, the product flavor and the fresh milk taste are investigated and evaluated in a scoring mode (10 points full), and the average points calculated finally are displayed in a table;

the overall rating was done after the test was tasted by 200 persons, and the overall product was rated for selection from likes, goodness, and commons, with the total number of persons selected for each item shown in the table.

TABLE 3 product evaluation Table

The present invention has been described in detail in order to enable those skilled in the art to understand the invention and to practice it, and it is not intended to limit the scope of the invention, and all equivalent changes and modifications made according to the spirit of the present invention should be covered by the present invention.