CN108112890B - Enzymolysis fermented bone meal and preparation method thereof - Google Patents
Enzymolysis fermented bone meal and preparation method thereof Download PDFInfo
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- 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
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/20—Meat products; Meat meal; Preparation or treatment thereof from offal, e.g. rinds, skins, marrow, tripes, feet, ears or snouts
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- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/40—Meat products; Meat meal; Preparation or treatment thereof containing additives
- A23L13/48—Addition of, or treatment with, enzymes
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
The invention belongs to the technical field of bone meal food, and particularly discloses enzymolysis fermented bone meal and a preparation method thereof. According to the invention, the neutral protease and the papain are combined to carry out enzymolysis on the fermented bovine bone meal for the first time, so that the quality is improved, the granular feeling of the bone meal is reduced, the bone meal is more smooth, the optimal conditions of enzymolysis and fermentation are determined, the bone meal is endowed with the fermentation flavor after being fermented by lactic acid bacteria, macromolecular substances are decomposed, the flavor is improved, the nutritional quality is improved, the added value of the product is high, and the value and the utilization rate of the bovine bone are improved.
Description
Technical Field
The invention belongs to the technical field of bone meal food, and particularly relates to enzymolysis fermented bone meal and a preparation method thereof.
Background
The 2017 version of Chinese beef market status investigation and development trend analysis report released by the Chinese industry investigation network considers that the United states, Brazil and China are the three countries before beef consumption. In 2016, the beef yield of China reaches 680 million tons, and in recent years, the beef yield of China tends to increase year by year. As the inner Mongolia of the first five pasturing areas in China, by the end of 2016 (11 months), cattle are out of stock in the whole area by 417.2 thousands of cattle, which is increased by 7.1 percent on a same scale. The beef yield is 71.3 ten thousand tons, and the beef yield is increased by 8.9 percent on the same scale. The weight of the cattle bone is 10% -20%, so the cattle bone yield in 2016 is about 7.9-17.8 ten thousand tons in China through rough statistics. Therefore, the ox bone yield is high in China. However, because the processing difficulty of the beef bones is high, the historical time for researching and developing the beef bones is short, the storage difficulty is high, beef cattle slaughter house enterprises have the influence of factors such as low consciousness and the like on the processing and utilization of the beef cattle byproducts, a large amount of beef bones are not effectively developed and utilized, and can only be added into feed to be used as a calcium supplement for livestock or processed into byproducts with poor mouthfeel, and the beef bones with high added values and high quality are few.
Compared with the domestic, the research on the bovine bone is more extensive abroad, for example, the bovine bone edible protein, the bone collagen and the animal gelatin are utilized in Germany, Britain, Italy, France and other countries; the superfine crushed bone powder is added into infant food, beef paste, beef cake, salted meat and other food in the United states.
Selenium is one of 15 elements essential to human body established by the health organization of the united nations, is a trace element which can not be synthesized by the human body and must be taken by food, has little demand and great effect, and is called as 'atomic bomb in trace element of human body' by the scientific community. Its six biological functions are: 1. antioxidation, anti-aging, 2, protecting and repairing cells, 3, improving the oxygen carrying capacity of erythrocytes, 4, improving the immunity of human bodies, 5, detoxifying and anti-pollution, 6, preventing canceration, and is called by world health organization: people should intake 50-250 micrograms of selenium every day like taking protein, and the supplement by natural organic selenium-rich food in food is most scientific. Because the vast areas in China are generally lack of selenium, the selenium supplement through the selenium-rich food is the most convenient method, so in recent years, the development and popularization of the selenium-rich food are one of the research hotspots of functional foods.
Therefore, the selenium-rich beef bone product with high added value is developed, the value and the utilization rate of the beef bone are improved, and the profits of beef enterprises in China can be further improved.
Disclosure of Invention
According to the enzymolysis fermented bone meal and the preparation method thereof provided by the invention, the enzymolysis fermented bone meal prepared by adopting a double-enzyme enzymolysis process has high added value of products, and the value and the utilization rate of the beef bone are improved.
The invention aims to provide enzymolysis fermented bone meal, which is obtained by preparing defatted fermented bone meal from a selenium-rich bovine bone raw material, performing enzymolysis by neutral protease and papain, fermenting by a mixed strain of lactobacillus bulgaricus and streptococcus thermophilus, and finally drying.
The invention also provides a preparation method of the enzymolysis and fermentation bone meal, which comprises the following steps:
s1, selecting selenium-rich bovine bone raw materials, and preparing defatted fermented bone powder;
s2, enzymolysis
Preparing 10g/100ml defatted bone powder solution, adjusting pH to 6-8, adding neutral protease and papain for enzymolysis, and inactivating enzyme at 90 deg.C for 20min to obtain enzymolysis fermented bone powder solution;
wherein the addition amount of the neutral protease is 10-14g/kg, the addition amount of the papain is 10-16g/kg, the enzymolysis temperature is 50-55 ℃, and the enzymolysis time is 4-6 h;
s3, fermentation
Adding glucose into the enzymolysis fermented bone meal solution, adjusting pH to 6-8, sterilizing, adding activated Lactobacillus bulgaricus seed solution and activated Streptococcus thermophilus seed solution, respectively, and fermenting for 24-30 hr to obtain fermented bone meal solution;
wherein the dosage ratio of the enzymolysis fermentation bone meal solution to the glucose is 100ml:5g, and the volume dosage ratio of the lactobacillus bulgaricus seed solution to the streptococcus thermophilus seed solution is 2: 1;
s4, evaporating the fermented bone meal solution to dryness, seasoning, and preparing the enzymolysis fermented bone meal.
Preferably, the preparation method of the enzymatic hydrolysis fermented bone meal comprises the following steps:
s11, fermentation bone meal rough preparation
Sequentially defatting selenium-rich ox bone raw material, removing bone marrow and bone oil, air drying, mashing, grinding into powder, and sieving to obtain crude fermented bone powder;
s12, defatting the fermented bone meal
Adding the crude fermented bone meal into 95% ethanol with a feed-liquid ratio of 1g to 15ml, defatting, and air drying to obtain defatted fermented bone meal.
Preferably, in the preparation method of the enzymatic hydrolysis fermented bone meal, the specific steps of S11 are as follows: sequentially decocting selenium-rich Os bovis Seu Bubali material with boiling water at normal pressure for 120min to remove fat, decocting at 0.1MPa for 40min to remove bone marrow and bone oil, air drying at 60-70 deg.C for 10 hr, mashing, grinding into powder, and sieving with 100 mesh sieve to obtain crude fermented bone powder.
Preferably, in the preparation method of the enzymolysis and fermentation bone meal, in S12, the degreasing time is 9h and the temperature is 45 ℃.
Preferably, in the preparation method of the enzymolysis and fermentation bone meal, in S2, the activity of neutral protease is 11036U/g, and the activity of papain is 45700U/g; the addition amount of the neutral protease is 13g/kg, and the addition amount of the papain is 15 g/kg; the temperature of enzymolysis is 55 ℃, and the time is 6 h.
Compared with the prior art, the enzymolysis and fermentation bone meal and the preparation method thereof have the following beneficial effects:
because the varieties of animal-derived selenium-rich products are few in the prior art, the invention selects the fermented bone meal prepared from the beef bones of the selenium-rich beef produced by cattle fed by selenium-rich feed by Kewanxin halal food Co., Ltd, Erdos city, Nemond, the selenium content is more than or equal to 300 micrograms/gram, the product belongs to the raw material of the selenium-rich beef bones, and the varieties of animal-derived selenium-rich products are increased.
The method has the advantages that neutral protease and papain are subjected to double-enzyme combined enzymolysis fermentation to ferment the bovine bone meal for the first time, the quality is improved, the granular feeling of the bone meal is reduced, the bone meal is more smooth, the optimal conditions of enzymolysis and fermentation are determined, the bone meal is endowed with the fermentation flavor after being fermented by lactic acid bacteria, macromolecular substances are decomposed, the flavor is improved, the nutritional quality is improved, the added value of the product is high, and the value and the utilization rate of the bovine bone are improved.
Drawings
FIG. 1 is the effect of the enzymolysis temperature on the degree of hydrolysis in a single factor experiment with neutral protease;
FIG. 2 is the effect of time on the degree of hydrolysis in a single factor experiment with neutral protease;
FIG. 3 is the effect of enzymatic pH on the degree of hydrolysis in a single factor experiment with neutral protease;
FIG. 4 is the effect of the amount of added enzymolysis enzyme on the degree of hydrolysis in a single factor experiment with neutral protease;
FIG. 5 is the effect of the enzymolysis temperature on the degree of hydrolysis in a single factor experiment of papain;
FIG. 6 is the effect of time on the degree of hydrolysis in a single factor experiment with papain;
FIG. 7 is the effect of enzymatic pH on hydrolysis in a single factor experiment with papain;
FIG. 8 shows the effect of the amount of enzyme on the degree of hydrolysis in a single element papain experiment.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.
Example 1
The embodiment 1 of the invention provides enzymolysis fermented bone meal, which is obtained by preparing defatted fermented bone meal from a selenium-rich bovine bone raw material, performing enzymolysis by using neutral protease and papain, fermenting by using a mixed strain of lactobacillus bulgaricus and streptococcus thermophilus, and drying. The preparation method of the enzymolysis and fermentation bone meal comprises the following steps:
s1, selecting selenium-rich bovine bone raw materials, and preparing defatted fermented bone powder
In the embodiment 1 of the invention, enzymolysis fermented bone meal of Nemontage Zhongke Wanxin Islamic food Limited is adopted, the company prepares the fermented bone meal by using the bovine bone of the selenium-rich beef produced by the cattle fed by the selenium-rich feed, and the selenium content of the fermented bone meal can reach 300 micrograms, thus the bone meal belongs to enzymolysis fermented bone meal. The preparation process of the defatted fermented bone meal comprises the following steps:
s11, fermentation bone meal rough preparation
Decocting selenium-rich Os bovis Seu Bubali material in boiling water at normal pressure for 120min to remove fat, decocting at 0.1MPa for 40min to remove bone marrow and bone oil, air drying at 60-70 deg.C for 10 hr, mashing, coarse grinding, fine grinding, grinding into powder, and sieving with 100 mesh sieve to obtain coarse fermented bone powder;
s12, defatting the fermented bone meal
Adding the crude fermented bone powder into 95 vol.% ethanol at a ratio of 1g to 15ml, stirring thoroughly with a magnetic stirrer (Tanskin, City, Zhengrong laboratory) for 9h at 45 deg.C for defatting, and air drying to obtain defatted fermented bone powder.
S2, enzymolysis
Accurately weighing the defatted and fermented bone meal in a beaker, adding distilled water to prepare 10g/100ml defatted bone meal solution, adjusting the pH to 6 by using a pH meter, adding neutral protease and papain, placing the mixture on a constant-temperature magnetic stirrer to stir for enzymolysis, and after the enzymolysis, putting enzymolysis liquid in a 90 ℃ water bath box to inactivate enzyme for 20min to obtain the enzymolysis and fermented bone meal solution.
Wherein the activity of the neutral protease is 11036U/g, and the activity of the papain is 45700U/g; the addition amount of the neutral protease is 13g/kg (taking the defatted bone powder solution as a measuring standard), and the addition amount of the papain is 15g/kg (taking the defatted bone powder solution as a measuring standard); the temperature of stirring enzymolysis is 55 ℃, and the time is 6 h.
S3, fermentation
S31 preparation of the Medium
The formula of the skim milk activation medium comprises: 100mL of skim milk is sterilized for 5min at 105 ℃ for later use.
The seed liquid is prepared by mixing the following components in percentage by mass: 15% of fermented bone meal, 5% of glucose, 10% of skim milk and 70% of distilled water; bottling, and sterilizing at 121 deg.C for 15 min.
S32 activation of lactic acid bacteria
Respectively inoculating Lactobacillus bulgaricus and Streptococcus thermophilus into skimmed milk culture medium, culturing at 40.5 deg.C for 6-10 hr, solidifying skimmed milk, placing in 4 deg.C refrigerator, and continuously activating for three times until thallus concentration of Lactobacillus bulgaricus and Streptococcus thermophilus reaches 108-109And (4) obtaining activated lactobacillus bulgaricus seed liquid and activated streptococcus thermophilus seed liquid respectively.
According to the actual dosage, the seed solution of S31 is used to respectively perform amplification culture on the activated lactobacillus bulgaricus and the activated streptococcus thermophilus until the required volumes of the lactobacillus bulgaricus seed solution and the streptococcus thermophilus seed solution are obtained, and the thallus concentrations of the lactobacillus bulgaricus seed solution and the streptococcus thermophilus seed solution reach 108-109One per ml.
S33, inoculation
Adding glucose into the enzymolysis and fermentation bone meal solution, adjusting the pH value to 6, sterilizing to obtain a fermentation culture medium, inoculating the activated lactobacillus bulgaricus seed solution and the activated streptococcus thermophilus seed solution into the fermentation culture medium respectively according to the inoculation amount of 4ml of seed solution per 100ml of culture medium, and fermenting for 24 hours to obtain the fermentation bone meal solution;
wherein the dosage ratio of the enzymolysis fermentation bone meal solution to the glucose is 100ml:5g, and the volume dosage ratio of the lactobacillus bulgaricus seed solution to the streptococcus thermophilus seed solution is 2: 1;
s4, evaporating the fermented bone powder solution to dryness by a conventional method (the water content in the evaporated fermented bone powder is less than or equal to 3%), seasoning (sodium citrate is added as a seasoning, the amount of the sodium citrate accounts for 0.1% of the mass of the evaporated fermented bone powder), preparing the enzymolysis fermented bone powder, and tabletting to prepare the chewable tablet of the enzymolysis fermented bone powder.
Example 2
The embodiment 2 of the invention provides enzymolysis fermented bone meal, which is obtained by preparing defatted fermented bone meal from a selenium-rich bovine bone raw material, performing enzymolysis by using neutral protease and papain, fermenting by using a mixed strain of lactobacillus bulgaricus and streptococcus thermophilus, and drying. The preparation method of the enzymolysis and fermentation bone meal comprises the following steps:
s1, selecting selenium-rich bovine bone raw materials, and preparing defatted fermented bone powder
In the embodiment 2 of the invention, enzymolysis fermented bone meal of Nemontage Zhongke Wanxin Islamic food Limited is adopted, the company prepares the fermented bone meal by using the bovine bone of the selenium-rich beef produced by the cattle fed by the selenium-rich feed, and the selenium content of the fermented bone meal can reach 300 micrograms, thus the bone meal belongs to enzymolysis fermented bone meal. The preparation process of the defatted fermented bone meal is the same as the operation steps of the embodiment 1.
S2, enzymolysis
Accurately weighing the defatted and fermented bone meal in a beaker, adding distilled water to prepare 10g/100ml defatted bone meal solution, adjusting the pH to 8 by using a pH meter, adding neutral protease and papain, placing the mixture on a constant-temperature magnetic stirrer to stir for enzymolysis, and after the enzymolysis, putting enzymolysis liquid in a 90 ℃ water bath box to inactivate enzyme for 20min to obtain the enzymolysis and fermented bone meal solution.
Wherein the activity of the neutral protease is 11036U/g, the activity of the papain is 45700U/g, the addition amount of the neutral protease is 13g/kg, and the addition amount of the papain is 15 g/kg; the temperature of stirring enzymolysis is 55 ℃, and the time is 6 h.
S3, fermentation
S31 preparation of the Medium
The formula of the skim milk activation medium comprises: 100mL of skim milk is sterilized for 5min at 105 ℃ for later use.
The seed liquid is prepared by mixing the following components in percentage by mass: 15% of fermented bone meal, 5% of glucose, 10% of skim milk and 70% of distilled water; bottling, and sterilizing at 121 deg.C for 15 min.
S32 activation of lactic acid bacteria
Respectively inoculating Lactobacillus bulgaricus and Streptococcus thermophilus into skimmed milk culture medium, culturing at 40.5 deg.C for 6-10 hr, solidifying skimmed milk, placing in 4 deg.C refrigerator, and continuously activating for three times until thallus concentration of Lactobacillus bulgaricus and Streptococcus thermophilus reaches 108-109And each/ml, respectively obtaining activated lactobacillus bulgaricus and activated streptococcus thermophilus.
S33, inoculation
Adding glucose into the enzymolysis and fermentation bone meal solution, adjusting the pH value to 8, sterilizing to obtain a fermentation culture medium, inoculating the activated lactobacillus bulgaricus seed solution and the activated streptococcus thermophilus seed solution into the fermentation culture medium according to the inoculation amount of 3ml of seed solution per 100ml of culture medium, and fermenting for 30 hours to obtain a fermentation bone meal solution;
wherein the dosage ratio of the enzymolysis fermentation bone meal solution to the glucose is 100ml:5g, and the volume dosage ratio of the lactobacillus bulgaricus seed solution to the streptococcus thermophilus seed solution is 2: 1;
s4, evaporating the fermented bone powder solution to dryness by a conventional method (the water content in the evaporated fermented bone powder is less than or equal to 3%), seasoning (sodium citrate is added as a seasoning, the amount of the sodium citrate accounts for 0.1% of the mass of the evaporated fermented bone powder), preparing the enzymolysis fermented bone powder, and tabletting to prepare the chewable tablet of the enzymolysis fermented bone powder.
Example 3
The embodiment 3 of the invention provides enzymolysis fermented bone meal, which is obtained by preparing defatted fermented bone meal from a selenium-rich bovine bone raw material, performing enzymolysis by using neutral protease and papain, fermenting by using a mixed strain of lactobacillus bulgaricus and streptococcus thermophilus, and drying. The preparation method of the enzymolysis and fermentation bone meal comprises the following steps:
s1, selecting selenium-rich bovine bone raw materials, and preparing defatted fermented bone powder
The operation was the same as in example 1;
s2, enzymolysis
The operation was the same as in example 1, except that the amount of neutral protease added was 10g/kg (based on the defatted bone powder solution), and the amount of papain was 16g/kg (based on the defatted bone powder solution); stirring and performing enzymolysis at 50 ℃ for 6 hours;
s3, fermentation
The operation was the same as in example 1.
Example 4
The embodiment 4 of the invention provides enzymolysis fermented bone meal, which is obtained by preparing defatted fermented bone meal from a selenium-rich bovine bone raw material, performing enzymolysis by using neutral protease and papain, fermenting by using a mixed strain of lactobacillus bulgaricus and streptococcus thermophilus, and drying. The preparation method of the enzymolysis and fermentation bone meal comprises the following steps:
s1, selecting selenium-rich bovine bone raw materials, and preparing defatted fermented bone powder
The operation was the same as in example 1;
s2, enzymolysis
The procedure was the same as in example 1, except that the amount of neutral protease added was 14g/kg (based on the defatted bone powder solution), and papain was 10g/kg (based on the defatted bone powder solution); stirring for enzymolysis at 53 deg.C for 5 h;
s3, fermentation
The operation was the same as in example 1.
Example 5
The test result of the zymohydrolysis process of the fermented bone meal is as follows:
1. test index measuring method
(1) Fat content calculation
Method for determining fat content by cable extraction method
X＝(M1-M2)×100
In the formula: x is the fat content in the sample, g/100 g;
m1-weight of filter paper bag before degreasing, g;
m2-weight of filter paper bag after degreasing, g;
(2) method for measuring degree of hydrolysis
The amino nitrogen is determined by formaldehyde titration, and the total nitrogen content is analyzed by a Kjeldahl azotometer
Degree of hydrolysis (%) - (% of total amino acids in hydrolysate/total nitrogen in defatted fermented bone powder) x 100%
(3) Determination of calcium conversion
Determination of calcium conversion by EDTA method
Calcium conversion (%) - (calcium content in fermentation broth centrifugation supernatant/calcium content in defatted fermented bone powder) x 100%
(4) Viable count determination
Viable cell counts were determined by a hemacytometer.
2. Results of the experiment
2.1 determination of Single-factor optimal conditions for neutral proteases
2.1.1 Effect of enzymolysis temperature on the degree of hydrolysis
The influence of the enzymolysis temperature on the hydrolysis degree in the single-factor experiment of the neutral protease is shown in fig. 1, and it can be known from fig. 1 that when the enzymolysis temperature is lower than 50 ℃, the hydrolysis degree is increased along with the increase of the temperature and is in positive correlation, because the activity of the enzyme is increased along with the increase of the temperature, and the hydrolyzed protein is gradually increased in unit time, so that the hydrolysis degree is increased; when the temperature reaches 50-60 ℃, the activity of the enzyme reaches the optimal state, the stability of the enzyme is higher, and the hydrolysis degree of the protein is highest. When the temperature is higher than 60 ℃, the structure of the enzyme is gradually changed, the stability of the enzyme begins to be reduced, the enzyme begins to be inactivated, and the activity of the enzyme gradually decreases along with the increase of the temperature. Therefore, in order to reduce energy consumption, we determined that the optimal temperature for the single-factor test of the neutral protease to enzymolyze the fermented bone meal solution is 50 ℃.
2.1.2 Effect of time on degree of hydrolysis
The influence of time on the degree of hydrolysis in the single-factor experiment with neutral protease is shown in FIG. 2, and it can be seen from FIG. 2 that the neutral protease does not react sufficiently with the defatted fermented bone meal and does not reach a state of saturation until the hydrolysis time does not reach 4 hours. Therefore, the enzyme and the substrate can fully react before, the hydrolysis speed of the enzyme is high at the stage, and the enzymolysis rate is increased quickly; however, as the enzymolysis reaction proceeds, the concentration of the reactant gradually decreases, and the enzymolysis products gradually increase to form more compounds, thereby hindering the proceeding of the enzymolysis reaction and gradually slowing down the enzymolysis reaction rate, so that the enzymolysis rate starts to decrease when the enzymolysis reaction proceeds for 4-6h, but the hydrolysis degree is continuously increased, and when the enzymolysis time reaches 6h, the substrate almost completely reacts and the hydrolysis degree hardly occurs. Therefore, we determined that the optimal time for the single-factor test neutral protease to enzymolyze the fermented bone meal solution is 6 h.
2.1.3 Effect of enzymatic hydrolysis pH on the degree of hydrolysis
The influence of the enzymolysis pH on the hydrolysis degree in the single-factor experiment of the neutral protease is shown in figure 3, and as can be seen from figure 3, when the enzymolysis pH value is between 6 and 8, the hydrolysis degree of the protein in the neutral protease enzymolysis fermentation bone meal is highest, and when the enzymolysis pH value is less than 6, the hydrolysis degree of the protein in the fermentation bone meal is increased along with the increase of the pH value, and the hydrolysis degree and the enzymolysis pH value form positive correlation; when the pH value of enzymolysis is more than 8, the hydrolysis degree of protein in the fermented bone meal is reduced along with the increase of the pH value, and the hydrolysis degree is in negative correlation with the pH value of the enzymolysis; when the enzymolysis pH is not between 6 and 8, the conformation of the neutral protease is changed, related groups of the active site of the enzyme molecule are dissociated, and the neutral protease and the substrate are in different dissociation states under the pH condition, so that the enzymolysis reaction is hindered, the combination of the enzyme and the substrate is influenced to generate a complex, and the activity of the enzyme is reduced. Therefore, the optimal enzymolysis pH of the neutral protease enzymolysis fermented bone meal solution is determined when the pH is 6-8 in the single-factor test.
2.1.4 Effect of the amount of enzymolysis enzyme added on the degree of hydrolysis
The influence of the amount of the added enzymolysis enzyme on the degree of hydrolysis in the single-factor experiment of the neutral protease is shown in FIG. 4. it can be seen from FIG. 4 that the degree of hydrolysis of the protein in the defatted fermented bone meal is continuously increased with the increasing amount of the added enzyme before the amount of the added enzyme is 10g/kg (10 g of neutral protease is added per kg of defatted bone meal solution, and the same measurement is adopted for different amounts of the added enzyme), and when the amount of the added enzyme reaches 10g/kg, the degree of hydrolysis of the substrate begins to become lower, and when the amount of the added enzyme reaches 12g/kg, the degree of hydrolysis of the enzyme hardly changes. This is because the degree of hydrolysis of the substrate has not reached a saturation state before 10g/kg, the degree of hydrolysis of the substrate has gradually reached a saturation state after the amount of the enzyme added reaches 10g/kg, and the degree of hydrolysis of the substrate has been completely saturated when the amount of the enzyme added reaches 12 g/kg. Therefore, the degree of hydrolysis does not change much after the addition amount of the enzyme reaches 12g/kg, and therefore, the optimal addition amount of the enzyme for carrying out enzymolysis on the defatted fermented bone meal solution by the neutral protease in the single-factor test is determined to be 12 g/kg.
2.2 determination of Single-factor optimal conditions for papain
2.2.1 Effect of enzymolysis temperature on the degree of hydrolysis
The influence of the enzymolysis temperature on the hydrolysis degree in the papain single-factor experiment is shown in fig. 5, and it can be seen from fig. 5 that when the temperature is lower than 60 ℃, the enzyme hydrolysis degree is lower due to lower enzyme activity and slower hydrolysis rate, and the hydrolysis degree is increased along with the increase of the enzymolysis temperature, and has positive correlation with the temperature of the enzyme; with the continuous rise of the temperature, when the enzymolysis temperature reaches 60 ℃, the hydrolysis degree of the papain reaches the maximum value, when the temperature gradually rises, the structure of the papain starts to change, and then, the hydrolysis degree starts to reduce. Therefore, the temperature of the papain for enzymolysis of the fermented bone meal in the single-factor test is determined to be 60 ℃.
2.2.2 Effect of enzymolysis time on the degree of hydrolysis
The influence of the enzymolysis time on the hydrolysis degree in the single-factor experiment of the papain is shown in fig. 6, and as can be seen from fig. 6, when the enzymolysis time is less than 4 hours, the substrate in the enzyme does not react completely, so that the hydrolysis degree increases along with the increase of the time, and when the enzymolysis time reaches 4 hours, the substrate reacts completely, and the hydrolysis degree of the enzyme begins to tend to a stable state. Therefore, the optimal enzymolysis time of the papain enzymolysis fermentation bone meal solution is 4 h. The degree of hydrolysis was low at the start of the reaction and increased with the increase in hydrolysis time, but after 4 hours, the rate of change of the degree of hydrolysis became small and thereafter the degree of hydrolysis tended to be constant. Therefore, the enzymolysis time of the papain is preferably 4 hours.
2.2.3 Effect of enzymatic hydrolysis pH on the degree of hydrolysis
The influence of the enzymolysis pH value on the hydrolysis degree in the papain single-factor experiment is shown in FIG. 7, and as can be seen from FIG. 7, when the pH value is 6, the hydrolysis degree is the highest; when the pH value is lower than 6, the hydrolysis degree of the papain is increased along with the increase of the pH value, when the enzymolysis pH value reaches 6, the hydrolysis degree reaches a peak value, and when the hydrolysis degree is lower than 6, the hydrolysis degree of the papain is decreased along with the increase of the pH value. When the pH value is higher or lower than 6, the combination of the papain and the protein in the fermented bone meal solution is blocked, so that the activity of the papain is reduced, even denatured and inactivated, and the pH value 6 is selected as the optimal enzymolysis pH value of the papain enzymolysis fermented bone meal solution.
2.2.4 Effect of the amount of enzymolysis enzyme added on the degree of hydrolysis
The effect of the amount of the added enzymolysis enzyme on the degree of hydrolysis in the single-factor papain experiment is shown in fig. 8, and it can be seen from fig. 8 that the degree of hydrolysis rapidly increases with the increase in the amount of the added enzyme before the amount of the added enzyme is 10g/kg, the increase in the degree of hydrolysis begins to slow when the amount of the added enzyme reaches 10g/kg, and the degree of hydrolysis reaches the maximum when the amount of the added enzyme is 14 g/kg. Therefore, the optimal addition amount of the papain enzymolysis and fermentation bone meal solution is selected as the enzyme addition amount of 15 g/kg.
2.3 screening of orthogonal assays under enzymatic conditions
Through the single factor tests of 2.1 and 2.2, the optimal conditions for obtaining the single factor of the neutral protease are as follows: enzymolysis at 50 deg.C for 6h, pH of 6-8, and enzyme addition amount of 12 g/kg. The optimal conditions of the single factor of the papain are as follows: the enzymolysis time is 4h at 60 ℃, the pH is 6, and the enzymolysis dosage is 15 g/kg. When the orthogonal test is designed, the enzymolysis time is set to 6h, the enzymolysis pH is set to a fixed value of 6, A is the enzymolysis temperature (DEG C) of neutral protease, B is the dosage (g/kg) of neutral protease, C is the enzymolysis temperature (DEG C) of papain, D is the dosage (g/kg) of papain, and the design scheme of the orthogonal test for enzymolysis of the fermented bone meal is shown in Table 1.
TABLE 1 fermentation bone meal enzymolysis orthogonal test design scheme
The data obtained after enzymatic hydrolysis according to the above protocol are shown in Table 2. As can be seen from Table 1, the influence sequence of each factor is B > D > A > C, namely, the enzymolysis temperature of neutral protease > the enzymolysis temperature of papain > the addition amount of neutral protease > the addition amount of papain. The most suitable process parameters are A2B2C3D1 in Table 1, namely the enzyme dosage of neutral protease is 13g/kg, the enzymolysis temperature of the neutral protease is 55 ℃, the enzyme dosage of papain is 15g/kg, and the enzymolysis temperature of the papain is 55 ℃.
Orthogonal experiments have shown that hydrolysis with two enzymes is more than a single enzyme, for example: under the conditions that the enzymolysis temperature of papain and neutral protease is 60 ℃, the enzymolysis time is 6h, the enzymolysis pH value is 6, and the enzyme addition amount is 13 g/kg; the degree of hydrolysis of neutral protease was 6.12%, the degree of hydrolysis of papain was 6.78%, and the degree of hydrolysis of the combination of the two enzymes was 10.21%. Thus, dual enzyme hydrolysis is somewhat preferred over single enzyme hydrolysis.
TABLE 2 orthogonal results of enzymatic hydrolysis test
Therefore, we determined that the optimal hydrolysis conditions for the fermented bone meal are: the concentration of the fermented bone meal is 10g/100 ml; double-enzyme hydrolysis; the hydrolysis temperature of the papain is 55 ℃, the enzyme addition amount is 15g/kg, the enzymolysis pH value is 6, and the enzymolysis time is 4 h; the hydrolysis temperature of the neutral protease is 55 ℃, the enzyme addition amount is 13g/kg, the enzymolysis pH value is 6, and the enzymolysis time is 6 h; the optimal fermentation conditions of the fermented bone meal are as follows: the concentration of the fermented bone meal is 10g/100 ml; the ratio of the strains of the lactobacillus bulgaricus and the streptococcus thermophilus is 2: 1; the optimal inoculation amount is 3 percent; the adding amount of glucose is 3%, and the fermentation time is 30 h; the hydrolysis rate of the double-enzyme hydrolysis under the hydrolysis condition and the fermentation condition is 12.58 percent; the conversion of calcium was 31.45%.
It should be noted that when the following claims refer to numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected, and since the steps and methods adopted are the same as those of the embodiments 1 to 4, the present invention describes the preferred embodiments 1 to 2 and the effects thereof in order to prevent redundancy, but once a person skilled in the art knows the basic inventive concept, other changes and modifications can be made to the embodiments. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (3)
1. A preparation method of enzymolysis fermentation bone meal is characterized by comprising the following steps:
s1, selecting selenium-rich bovine bone raw materials, and preparing defatted fermented bone powder;
the process for preparing the defatted fermented bone meal comprises the following steps:
s11, fermentation bone meal rough preparation
Decocting selenium-rich Os bovis Seu Bubali material in boiling water at normal pressure for 120min to remove fat, decocting at 0.1MPa for 40min, removing bone marrow and bone oil, air drying at 60-70 deg.C for 10 hr, mashing, grinding into powder, and sieving with 100 mesh sieve to obtain crude fermented bone powder;
s12, defatting the fermented bone meal
Adding the crude fermented bone meal into 95% ethanol with a feed-liquid ratio of 1g to 15ml, degreasing, and air-drying to obtain defatted fermented bone meal;
s2, enzymolysis
Preparing 10g/100ml defatted bone powder solution, adjusting pH to 6-8, adding neutral protease and papain for enzymolysis, and inactivating enzyme at 90 deg.C for 20min to obtain enzymolysis fermented bone powder solution;
wherein the addition amount of the neutral protease is 10-14g/kg, the addition amount of the papain is 10-16g/kg, the enzymolysis temperature is 50-55 ℃, and the enzymolysis time is 4-6 h;
s3, fermentation
Adding glucose into the enzymolysis fermented bone meal solution, adjusting pH to 6-8, sterilizing, adding activated Lactobacillus bulgaricus seed solution and activated Streptococcus thermophilus seed solution, respectively, and fermenting for 24-30 hr to obtain fermented bone meal solution;
wherein the dosage ratio of the enzymolysis fermentation bone meal solution to the glucose is 100ml:5g, and the volume dosage ratio of the lactobacillus bulgaricus seed solution to the streptococcus thermophilus seed solution is 2: 1;
s4, evaporating the fermented bone meal solution to dryness, seasoning, and preparing the enzymolysis fermented bone meal.
2. The method for preparing enzymatically hydrolyzed and fermented bone meal according to claim 1, wherein the defatting time in S12 is 9 hours and the temperature is 45 ℃.
3. The method for preparing zymolytic fermented bone meal according to claim 1, wherein in S2, the activity of neutral protease is 11036U/g, and the activity of papain is 45700U/g; the addition amount of the neutral protease is 13g/kg, and the addition amount of the papain is 15 g/kg; the temperature of enzymolysis is 55 ℃, and the time is 6 h.
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