CN114246246A - Desensitization method for separating whey protein containing lactoferrin - Google Patents
Desensitization method for separating whey protein containing lactoferrin Download PDFInfo
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- CN114246246A CN114246246A CN202111646844.XA CN202111646844A CN114246246A CN 114246246 A CN114246246 A CN 114246246A CN 202111646844 A CN202111646844 A CN 202111646844A CN 114246246 A CN114246246 A CN 114246246A
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
- A23J3/08—Dairy proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/123—Bulgaricus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/515—Animalis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides a desensitization method of isolated whey protein containing lactoferrin, which comprises the following steps: s1, mixing the separated whey protein powder and ferrous salt by using normal saline, adding hydrolyzed casein, homogenizing, and sterilizing to obtain solution A; s2, mixing beef extract, peptone, yeast extract powder, hydrolyzed casein, glucose, 0.5% of sodium acetate, diammonium citrate, Tween, magnesium sulfate, manganese sulfate and L-cysteine hydrochloride in water, adjusting the pH value to 6-6.5, and sterilizing II to obtain a solution B; s3, mixing the solution A and the solution B to obtain a solution C; inoculating animal bifidobacterium and lactobacillus bulgaricus into the solution C, fermenting and drying.
Description
Technical Field
The invention belongs to the field of fermentation, and particularly relates to a desensitization method for separating whey protein containing lactoferrin.
Background
Whey protein is a general term for various globular proteins separated from whey, and the protein content of the separated whey protein is 90% or more, which is called "king of protein" because of many advantages such as high absorption rate and most reasonable amino acid composition. The whey protein is easy to digest, has the characteristics of high biological value, high digestibility, high protein efficiency ratio and high utilization rate, is a fine product in protein and the like, and is one of the accepted high-quality protein supplements for human bodies.
The nutritional value of whey protein:
first, among various proteins, the nutritional value of whey protein is the highest. In general, proteins having complete types and contents of essential amino acids and providing human needs can be called high-quality proteins, also called complete proteins. It contains 8 kinds of amino acids essential for human body, and has reasonable proportion and human body requirement ratio, and is essential matter for human body growth, development, senility resisting and other life activities.
Secondly, whey protein is easier to digest and absorb, and the milk contains 60% of whey protein and 40% of casein, so that the feces of the infants who drink the breast milk are softer and less in amount. In addition, whey is rich in cysteine and methionine, which maintain antioxidant levels in humans. Many experimental studies have also demonstrated that administration of whey protein concentrates can promote humoral and cellular immunity, stimulate the immune system of the human body, and prevent the development of chemically induced cancers. Therefore, the whey protein is a very good protein for enhancing immunity.
Thirdly, whey protein is low in fat and lactose, but it contains beta-lactoglobulin, alpha-lactalbumin, immunoglobulin, and other active ingredients. It is these active ingredients that provide whey protein with many health benefits to the human body and therefore it is considered to be one of the high quality protein sources required by the human body.
Since the isolated whey protein has a high protein content, it is highly allergenic to human bodies. According to survey, the milk allergy rate of children under 2 years old can reach 2.0%. Among the isolated whey proteins, the major allergenic proteins are casein, β -lactoglobulin and α -lactalbumin. Besides the proteins causing allergy, lactoferrin, immunoglobulin and the like are safe to human bodies and have high nutritional values.
Lactoferrin is an iron-binding glycoprotein with a molecular weight of 80kDa and belongs to the transferrin family. The amino acid sequence homology of bovine lactoferrin and human lactoferrin reaches 69%, and the bovine lactoferrin and human lactoferrin have similar biological activity, so that the bovine lactoferrin and human lactoferrin are one of the most easily available lactoferrin sources at present. Lactoferrin is high in colostrum and milk, and low in tear, saliva, semen, nasal and bronchial secretions, bile and gastrointestinal fluids, etc. In addition, lactoferrin is also a component of neutrophils. Lactoferrin not only participates in the transport of iron, but also has the powerful biological functions of broad-spectrum antibiosis, antioxidation, anticancer, immune system regulation and the like.
If one could find a way to reduce the sensitization of the proteins in the whey protein isolate while retaining the activity of other proteins that do not cause sensitization, the safety of eating a high protein nutritional product such as whey protein isolate could be greatly improved.
Most of the currently used protein desensitization methods achieve the purpose of desensitization by protein modification, and the commonly used methods are as follows:
1. physical methods are classified into thermal processing, ultra-high pressure, irradiation, ultrasonic treatment, and the like. The methods have high requirements on equipment and high energy consumption, and cannot ensure that the original sensory quality and nutritional ingredients of the protein are not damaged;
2. the chemical method mainly focuses on glycosylation and application thereof, and the emulsification capacity, oxidation resistance and thermal stability of the protein modified by glycosylation are obviously enhanced. But glycosylation is generally accompanied by the production of byproducts such as acrylamide, which causes central nervous system disorders and liver damage, posing a threat to human health;
3. the enzymolysis method, which utilizes specific protease to carry out enzymolysis on the protein causing allergy, can achieve the effect of desensitization of the protein. However, these enzymes have specificity, require different enzyme combinations for treatment, require different reaction temperatures and different reaction times, and have insufficient enzymatic hydrolysis degree, so that the risk of incomplete treatment of the allergenic protein still exists. Which enzyme combination, enzymolysis time, enzymolysis temperature, etc. need to be continuously searched, and the treatment is more complicated.
Disclosure of Invention
The invention aims to provide a desensitization method of an isolated whey protein containing lactoferrin. The method utilizes microbial fermentation to decompose the sensitized protein in the separated whey protein, thereby achieving the purpose of desensitization.
The purpose of the invention is realized by the following technical scheme.
A method of desensitizing isolated whey protein containing lactoferrin comprising the steps of:
s1, mixing whey protein powder and ferrous salt by using normal saline, adding hydrolyzed casein, homogenizing, and sterilizing I to obtain a solution A;
s2, mixing beef extract, peptone, yeast extract powder, hydrolyzed casein, glucose, sodium acetate, diammonium citrate, tween, magnesium sulfate, manganese sulfate and L-cysteine hydrochloride in water, adjusting the pH value to 6-6.5, and sterilizing II to obtain a solution B;
s3, mixing the solution A and the solution B to obtain a solution C; inoculating animal bifidobacterium and lactobacillus bulgaricus into the solution C, fermenting and drying.
The ferrous salt comprises ferrous nitrate, ferrous sulfate or ferrous chloride.
In the solution A, the content of the isolated whey protein powder is 60 wt%;
in the solution A, the content of ferrous salt is 0.5 wt%;
the content of hydrolyzed casein in the solution A was 1 wt%.
The sterilization I is high-temperature instantaneous sterilization.
The temperature of the sterilization II is 120-125 ℃;
the time for sterilizing II is 15 min.
The inoculation amount of the animal bifidobacterium is 3 percent;
the inoculation amount of the lactobacillus bulgaricus is 2 percent.
The temperature of the fermentation is 37 ℃;
the drying comprises spray drying.
The concentration of the solution B is 80%.
In the solution C, the concentration of beef extract is 0.5%, the concentration of peptone is 0.5%, the concentration of yeast extract powder is 0.5%, the concentration of glucose is 2%, the concentration of sodium acetate is.5%, the concentration of diammonium citrate is 0.2%, the concentration of Tween 80 is 0.1%, the concentration of magnesium sulfate is 0.058%, the concentration of manganese sulfate is 0.028%, and the concentration of L-cysteine hydrochloride is 0.05%.
Compared with the prior art, the invention has the following beneficial effects:
the invention can completely retain the activity of the lactoferrin in the fermentation process, so that the edible safety of the fermented separated whey protein is improved, and the fermented separated whey protein contains the active lactoferrin.
The invention uses the method of microbial fermentation to ferment the separated whey protein, and the lactobacillus used for fermentation can play the roles of resisting anaphylactic reaction and relieving anaphylactic symptoms.
Drawings
Figure 1 shows a flow chart of the desensitization method of the present invention.
Figure 2 shows the rate of change of the antigens beta-lactoglobulin and alpha-lactalbumin before and after fermentation in the isolated whey proteins of the examples.
Detailed Description
The invention provides a desensitization method of isolated whey protein containing lactoferrin. The principle of the invention is as follows: the probiotics can decompose the protein in the surrounding environment during fermentation, in order to prevent the lactoferrin from being decomposed, the lactoferrin is firstly enabled to reach an iron saturation state, and the lactoferrin can be stably preserved in the process of passing through the fermentation. Because the binding capacity of the lactoferrin and iron is strongest under the condition of neutral pH, the formed lactoferrin in an iron saturation state has stable structure and strongest enzymolysis resistance and bacteriostasis effects. After fermentation is completed, pH is reduced, the binding capacity of lactoferrin and iron ions is weakened, the bacteriostatic capacity is weakened, the growth of probiotics is not interfered, meanwhile, lactoferrin is used as a bifidus factor and can promote the proliferation of bifidobacteria, and free iron ions can participate in the cell membrane synthesis of the probiotics and promote the proliferation of the probiotics.
The principle of broad-spectrum antibacterial effect of lactoferrin is that lactoferrin can take iron ions from bacterial cell membrane to break cell membrane. The ability of lactoferrin to bind iron ions is greatly affected by pH and is a reversible process. The iron binding capacity is strongest under the neutral condition, the bacteriostatic ability is strongest, and when the pH value is reduced, the iron binding capacity is weakened, and the bacteriostatic ability is reduced.
The present invention enables desensitization of isolated whey proteins because an imbalance in the intestinal flora is also an important cause of food allergy. Because the activities of lactobacillus and bifidobacterium in the intestinal tracts of allergic infants are obviously lower than those of non-allergic infants, the physiological environment of the intestinal tracts has important influence on the occurrence of allergic reaction, so that the reduction of the protein allergy by a microbial fermentation method is a safer method at present and is also beneficial to industrial production. However, since various enzymes are secreted during the fermentation of microorganisms to decompose the proteins in the culture environment indiscriminately for the proliferation of the microorganisms, the invention provides a method for retaining the proteins which do not cause allergic reactions, namely lactoferrin in the separated whey proteins, and the lactoferrin is in an iron saturation state, is stable in structure and is not easily decomposed by the microorganisms in a mode of combining iron ions with the lactoferrin before the fermentation process begins. As the fermentation progresses, the pH value of the culture medium is reduced, the binding capacity of lactoferrin and iron ions is reduced, the iron ions can be recovered to be in an iron unsaturated state, and meanwhile, the liberated iron ions can promote the growth and proliferation of animal bifidobacteria and lactobacillus bulgaricus. The method can promote the growth and reproduction of probiotics, decompose and separate the allergic protein in the whey protein, and maintain the biological activity of the lactoferrin.
Specifically, 60% of separated whey protein powder and 0.5% of ferrous ions are added into the solution A, and the ferrous ions react with lactoferrin in the whey protein powder, so that the lactoferrin forms a stable structure and is not easily decomposed by microorganisms in the subsequent fermentation process. And the lactoferrin can promote the growth of animal bifidobacterium and is a bifidus factor.
Specifically, the hydrolyzed casein with the concentration of 1% is added into the solution A because free amino acids and small peptides in the solution A are not enough to support the growth of the probiotics, and the addition of the hydrolyzed casein can improve the growth rate of the probiotics.
In particular, the bifidobacterium animalis is adopted as the fermentation strain in the invention because the bifidobacterium animalis (HX-BA21) is one of the dominant bacteria in intestinal tracts of human beings and many mammals, and the lactose is fermented to generate galactose, so that the utilization rate of milk products of lactose intolerance patients can be improved. Promote the digestion and absorption of casein and milk protein, decompose lactose and improve lactose intolerance. The casein enzyme secreted by the animal bifidobacterium has high activity, and the casein utilization rate of one of the allergenic proteins in the separated whey protein is high.
Specifically, the lactobacillus bulgaricus is adopted as the fermentation strain in the invention because the lactobacillus bulgaricus (HX-LB66) is a lactic acid bacterium commonly used for producing fermented foods. Besides producing lactic acid, the protein hydrolysate has a good protein hydrolysis system and has strong capability of decomposing beta-lactoglobulin and alpha-lactalbumin.
Concretely, solution A after the homogeneity can not the solid-liquid layering, can let the thallus adhesion on the suspended particles, and the fixed point fermentation of stewing need not use agitating unit in fermentation process, can avoid increasing the circulation of gases such as oxygen among the stirring process, easy operation to more do benefit to and create the low oxygen environment, the growth of the probiotic of being convenient for.
Examples
Adding whey protein powder into normal saline, wherein the weight of the whey protein powder is 60% and the weight of the normal saline is 40% by weight. Adding 0.5% ferrous salt into the whey protein normal saline solution, mixing, standing for 0.5 h, adding 1% hydrolyzed casein, and mixing uniformly. Then using an ultrahigh temperature instant sterilization method to sterilize at 130-150 ℃ for 3-5 seconds to obtain solution A.
Preparing aqueous solution with concentration of 80% from beef extract, peptone, yeast extract powder, glucose, sodium acetate, diammonium citrate, tween 80, magnesium sulfate, manganese sulfate and L-cysteine hydrochloride, and adjusting pH to 6.0-6.5. In the base, the content of beef extract is 0.625%, the content of peptone is 0.625%, the content of yeast extract powder is 0.625%, the content of glucose is 2.5%, the content of sodium acetate is 0.625%, the content of diammonium citrate is 0.25%, the content of Tween 80 is 0.125%, the content of magnesium sulfate is 0.0725%, the content of manganese sulfate is 0.035%, and the content of L-cysteine hydrochloride is 0.0625%. The aqueous solution was subjected to moist heat sterilization at 121 ℃ for 15 minutes, and cooled to obtain solution B.
And (3) uniformly mixing the solution A and the solution B under the aseptic operation condition to obtain a solution C.
Inoculating animal bifidobacteria and lactobacillus bulgaricus into the solution C, and stirring uniformly. The inoculation amount of the animal bifidobacterium is 3 percent, and the inoculation amount of the lactobacillus bulgaricus is 2 percent.
Fermenting the inoculated solution C at 37 deg.C, standing for 10 hr, and spray drying. The air inlet temperature of the spray drying is not more than 110 ℃, and the air outlet temperature is about 50 ℃.
Examples the antigenicity of the two allergens beta-lactoglobulin and alpha-lactalbumin residues in the whey protein fermented product. The quantitative determination is carried out by sandwich enzyme-linked immunosorbent assay (ELISA). As shown in fig. 2, beta-lactoglobulin and alpha-lactalbumin in isolated whey protein are considered as major allergens causing pediatric allergy. As shown in the figure, the antigenicity of the two proteins can be greatly reduced through fermentation, and the edible safety is improved.
Detecting the content of lactoferrin by a high performance liquid chromatography method, and comparing data before fermentation. The specific values are as follows:
| before fermentation | After fermentation | |
| Lactoferrin content (%) | 1.24％ | 1.08％ |
From the above table, it can be seen that the lactoferrin content before fermentation is reduced by only 0.16% compared to after fermentation, and the loss rate is very low. This fermentation process was demonstrated to break down the allergenic proteins without loss of lactoferrin.
Claims (9)
1. A method of desensitizing isolated whey protein containing lactoferrin, comprising the steps of:
s1, mixing the separated whey protein powder and ferrous salt by using normal saline, adding hydrolyzed casein, homogenizing, and sterilizing to obtain solution A;
s2, mixing beef extract, peptone, yeast extract powder, hydrolyzed casein, glucose, sodium acetate, diammonium citrate, Tween, magnesium sulfate, manganese sulfate and L-cysteine hydrochloride in water, adjusting the pH value to 6-6.5, and sterilizing II to obtain a solution B;
s3, mixing the solution A and the solution B to obtain a solution C; inoculating animal bifidobacterium and lactobacillus bulgaricus into the solution C, fermenting and drying.
2. A method of desensitizing an isolated whey protein containing lactoferrin according to claim 1, wherein:
the ferrous salt comprises ferrous nitrate, ferrous sulfate or ferrous chloride.
3. A method of desensitizing an isolated whey protein containing lactoferrin according to claim 1, wherein:
in the solution A, the content of the isolated whey protein powder is 60 wt%;
in the solution A, the content of ferrous salt is 0.5 wt%;
the content of hydrolyzed casein in the solution A was 1 wt%.
4. A method of desensitizing an isolated whey protein containing lactoferrin according to claim 1, wherein:
the sterilization I is high-temperature instant sterilization.
5. A method of desensitizing an isolated whey protein containing lactoferrin according to claim 1, wherein:
the temperature of the sterilization II is 120-125 ℃;
the time for sterilization II is 15 min.
6. A method of desensitizing an isolated whey protein containing lactoferrin according to claim 1, wherein:
the inoculation amount of the animal bifidobacterium is 3 percent;
the inoculation amount of the lactobacillus bulgaricus is 2 percent.
7. A method of desensitizing an isolated whey protein containing lactoferrin according to claim 1, wherein:
the temperature of the fermentation is 37 ℃;
the drying comprises spray drying.
8. A method of desensitizing an isolated whey protein containing lactoferrin according to claim 1, wherein:
the concentration of the solution B is 80%.
9. A method of desensitizing an isolated whey protein containing lactoferrin according to claim 1, wherein:
in the solution B, the concentration of beef extract is 0.5%, the concentration of peptone is 0.5%, the concentration of yeast extract powder is 0.5%, the concentration of glucose is 2%, the concentration of sodium acetate is 0.5%, the concentration of diammonium citrate is 0.2%, the concentration of Tween 80 is 0.1%, the concentration of magnesium sulfate is 0.058%, the concentration of manganese sulfate is 0.028%, and the concentration of L-cysteine hydrochloride is 0.05%.
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