CN113142300A - Method for preparing horse milk product with ACE (angiotensin converting enzyme) inhibitory activity by combining yeast fermentation and enzymolysis - Google Patents
Method for preparing horse milk product with ACE (angiotensin converting enzyme) inhibitory activity by combining yeast fermentation and enzymolysis Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
- A23C9/1209—Proteolytic or milk coagulating enzymes, e.g. trypsine
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
Abstract
The invention discloses a method for preparing horse milk products with ACE inhibitory activity by combining yeast fermentation and enzymolysis, which belongs to the field of functional fermented foods, and is characterized in that sterilized fresh horse milk is fermented by yeast and then is subjected to protease enzymolysis to prepare the horse milk products with ACE inhibitory activity; the method is simple, the production efficiency is high, the ACE inhibitory activity of the horse milk product can reach more than 70%, and compared with clinical ACE inhibitor medicines, the horse milk product has the advantages of no toxic or side effect, high edible safety, easiness in digestion, capability of effectively inhibiting the ACE activity and the like.
Description
Technical Field
The invention relates to a horse milk product with ACE inhibitory activity prepared by combining yeast fermentation and enzymolysis and a production method thereof, belonging to the technical field of fermented foods.
Background
Hypertension is the most common chronic disease in recent years, is a global public health problem, and is also a key risk factor for inducing various complications. Angiotensin Converting Enzyme (ACE) plays an important role in the development and progression of hypertension as a core enzyme of the renin-Angiotensin System (RAS) and Kallikrein-Kinin System (KKS), and thus ACE inhibitors are considered as one of the best antihypertensive drugs. The ACE inhibitor medicament developed clinically at present has a remarkable antihypertensive effect, but has adverse reactions of different degrees. In recent years, milk ACE inhibitors have become a research focus of people in recent years due to the characteristics of high edible safety, capability of preventing and controlling hypertension, no toxic or side effect and the like.
At present, the preparation method of the milk-derived ACE inhibitor mainly comprises microbial fermentation and protease hydrolysis. The protease hydrolysis method has the advantages of safety, no toxic or side effect, mild hydrolysis condition, easy control and the like. The microbial fermentation method is a method for releasing functional polypeptide by hydrolyzing raw material protein by protease and peptidase generated in the fermentation process of microorganisms, and has the advantages of wide microbial source, multiple protease types, high yield, low cost and the like. At present, the research on preparing dairy products with ACE inhibitory activity by combining a protease hydrolysis method and a fermentation method is less, the fermented milk with ACE inhibitory activity mainly takes lactic acid bacteria fermented milk and lactic acid bacteria and saccharomycetes co-fermented milk as main materials, the research on horse milk is less, and the research on the horse milk fermented by saccharomycetes alone is almost not carried out. Researches show that the horse milk has the advantages of high nutritive value, rich protein content, high content of polyunsaturated fatty acid and vitamin, easy digestion and the like, and has important nutritive and therapeutic properties.
Disclosure of Invention
In order to prepare the horse milk with ACE inhibitory activity, the invention takes the fresh horse milk as a raw material, uses saccharomycetes to ferment the fresh horse milk to prepare fermented horse milk, and then adds protease to hydrolyze to obtain the horse milk product with ACE inhibitory activity.
The invention proves that the ACE inhibitory activity of the horse milk product can be improved by combining the fermentation method and the enzymolysis method compared with the single method, and a theoretical basis is provided for disclosing the biological mechanism of lowering blood pressure of the horse milk product and the probiotic effect of the microzyme.
The production process of the invention is as follows:
(1) activating strains: inoculating yeast into YPD liquid culture medium, culturing at 20-35 deg.C and 100-300 rpm for 12-48 hr, continuously proliferating for 2-4 generations, with the inoculum size being 0.5-3% by volume, and the viable count of activated bacteria liquid being not less than 108CFU/mL for standby;
(2) filtering or centrifuging fresh horse milk to purify the milk, and sterilizing at 60-150 ℃ for 2 s-30 min;
(3) inoculating activated yeast when horse milk is cooled to 20-40 ℃, wherein the inoculation amount is 0.5-6%, and the viable count in the horse milk after inoculation is 106～109CFU/mL；
(4) Fermenting the horse milk under the following conditions: fermenting for 4-80 h at 20-40 ℃ and with stirring speed of 5-200 rpm and ventilation of 10-600 vvm;
(5) hydrolyzing the fermented horse milk by using protease for 0.5 to 6 hours at the temperature of 28 to 50 ℃; the mass ratio of the protease to the fermented horse milk is 2-8%.
(6) The ACE inhibitory activity of horse milk products is determined by an HPLC method, which comprises the following steps: the reaction is carried out in a centrifugal tube of 1.5mL, 100 mu L of 5mmol/L of Hippuryl-histidyl-leucine (N-Hippuryl-His-Leu hydrate, HHL) solution is added, then 40 mu L of hydrolysate obtained in the step (5) is added for incubation at 37 ℃ for 5-10 min, then 40 mu L of LACE crude extract (extract containing ACE extracted from pig lung) is added for reaction at 37 ℃ for 30min, boiling is carried out immediately after the reaction is finished for 10min to stop the reaction, 400 mu L of deionized water is added for 10000g, centrifugation is carried out for 5min to remove precipitates, supernatant is absorbed, a 0.45 mu m filter head is used for filtering into a high-performance liquid phase bottle, and the sample injection volume is 20 mu L. The HPLC detection conditions are as follows: and (3) analyzing the column: kromasil C18150X 4.6 mm 5 μm E102427; mobile phase: 25% acetonitrile +75% water, 0.1% trifluoroacetic acid (TFA); column temperature: 30 ℃; flow rate: 1 mL/min; elution was monitored at 228 nm; the end time is 10 min. The ACE inhibitory activity of the fermented milk was calculated according to the following formula.
In the formula:
a: represents the amount of Hippuric Acid (HA) produced by the HHL and ACE reaction when the sample was replaced with buffer;
b: represents the amount of Hippuric Acid (HA) produced by the HHL and ACE reaction when the sample is added;
the yeast is saccharomyces cerevisiae (A) with single sporeKazachstania unispora) Kluyveromyces lactis (A), (B), (C)Kluyveromyces lactis) Kluyveromyces marxianus (K.marxianus)Kluyveromyces marxianus) Saccharomyces cerevisiae (A)Saccharomyces cerevisiae) Fermented Pichia pastoris (A)Pichia fermentans) Dekkera heteroclides (D.heteroclides) ((B))Dekkera anomala) One or more of the above-mentioned strains are conventional commercially available strains.
The protease is one or more of trypsin, pepsin, neutral protease, alkaline protease, elastase, bromelain, papain, compound protease, flavourzyme and the like, and is conventional commercially available protease.
The invention has the following advantages and positive effects:
1. the invention proves that the horse milk can be used as a high-quality milk source for preparing the fermented milk with the ACE inhibitory activity, and further provides a basis for disclosing the biological mechanism of lowering blood pressure of the horse milk product;
2. the invention provides theoretical basis for the probiotic effect of the yeast, and the yeast is taken as a strain for preparing the horse milk product with ACE inhibitory activity, so that the yeast is proved to have proteolytic activity and can be taken as a fermentation strain for preparing the horse milk product with ACE inhibitory activity;
3. the horse milk product with ACE inhibitory activity is prepared by a method combining yeast fermentation and protease hydrolysis, and the ACE inhibitory activity of the horse milk product can be improved by combining the two methods compared with a single method;
4. the production method for preparing the ACE inhibitory activity horse milk product has the advantages of simple process, high production efficiency and the like;
5. the horse milk product prepared by the invention has the characteristics of high edible safety, good stability and the like.
Drawings
FIG. 1 is the results of ACE inhibitory activity of horse milk samples treated in different ways according to example 1;
FIG. 2 shows the results of ACE inhibitory activity of horse milk samples treated in different ways according to example 2;
FIG. 3 is the results of ACE inhibitory activity of horse milk samples treated in different ways according to example 3;
FIG. 4 shows the results of ACE inhibitory activity of horse milk samples treated in different ways in example 4;
FIG. 5 shows the results of ACE inhibitory activity of horse milk samples treated in different ways according to example 5;
in the above figures, lower case letters indicate differential significance, the same letter indicates no significant difference (P < 0.05), and different letters indicate significant difference (P > 0.05); g1: fermenting horse milk with yeast; g2: hydrolyzing horse milk product with protease; g3: horse milk product prepared by combining yeast fermentation and protease hydrolysis.
Detailed Description
The technical scheme of the invention is further described in detail by the following examples, but the content of the invention is not limited to the examples, the methods in the examples are all conventional methods unless otherwise specified, and the materials, reagents and the like used in the examples are obtained from commercial approaches unless otherwise specified;
the details of the species used in the examples are as follows:
saccharomyces cerevisiae monospora (Kazachstania unispora) Purchased from the american type culture institute, strain number ATCC 10612; kluyveromyces marxianus (K.), (Kluyveromyces marxianus) And Saccharomyces cerevisiae (Saccharomyces cerevisiae) The strains are purchased from China center for culture Collection of industrial microorganisms, and the numbers of the strains are CICC 32423 and CICC 32415 respectively.
The specific detection methods in the following examples are as follows:
the method for measuring the viable count of the yeast comprises the following steps: counting is carried out according to the national standard GB 4789.15-2016 food safety national standard food microbiology test mould and yeast count, and an average value is taken after each product is repeated for 3 times.
Example 1: the production method of the horse milk product of the embodiment is as follows:
(1) inoculating Saccharomyces cerevisiae to YPD liquid culture medium, culturing at 30 deg.C and 200rpm for 12 hr, continuously proliferating for 2 generations, with the inoculum size being 1.83%, and the viable count of activated bacteria liquid being not less than 108CFU/mL for standby;
(2) filtering fresh horse milk, and sterilizing at 95 deg.C for 15 min;
(3) inoculating activated Saccharomyces cerevisiae when horse milk is cooled to 35 deg.C, wherein the inoculation amount is 4%, and the viable count in horse milk after inoculation is 106～9CFU/mL；
(4) Fermenting the horse milk under the following conditions: fermenting for 60h at 35 ℃ under the conditions of stirring speed of 200rpm and ventilation capacity of 25 vvm;
(5) hydrolyzing the fermented horse milk for 2 hours at 35 ℃ by using trypsin, wherein the mass ratio of the protease to the fermented horse milk is 6%;
(6) three samples of single-spore Saccharomyces cerevisiae fermented milk, trypsin hydrolyzed horse milk product, and horse milk product combining single-spore Saccharomyces cerevisiae fermentation and trypsin hydrolysis were tested for ACE inhibitory activity by HPLC.
The viable count of the yeast in this example is shown in table 1, and the ACE inhibition activities of the three samples in this example are shown in fig. 1, and it is known from the results that the combination of saccharomyces cerevisiae fermentation and trypsin hydrolysis can significantly improve the ACE inhibition rate of the horse milk product, and compared with the saccharomyces cerevisiae fermented milk alone, the ACE inhibition rate is improved by 42.77%, and compared with the horse milk hydrolyzed by trypsin alone, the ACE inhibition rate is improved by 26.4%. Therefore, the ACE inhibition rate of the horse milk product can be obviously improved by combining single-spore saccharomyces cerevisiae fermentation and trypsin hydrolysis;
TABLE 1 viable count of yeasts in horse milk after inoculation
| Sample (I) | Number of viable bacteria |
| Activated bacterial liquid | 2.66×109CFU/mL |
| Horse milk after inoculation | 1.8×108CFU/mL |
Example 2: the production method of the horse milk product of the embodiment is as follows:
(1) inoculating Saccharomyces cerevisiae to YPD liquid culture medium, culturing at 27 deg.C and 200rpm for 15h, continuously proliferating for 3 generations, with the inoculum size being 2.5% by volume, and the viable count of activated bacteria liquid being not less than 108CFU/mL for standby;
(2) filtering fresh horse milk, and sterilizing at 120 deg.C for 30 s;
(3) inoculating 3.5% of Saccharomyces cerevisiae with the horse milk cooled to 30 deg.C, and the number of viable bacteria in the horse milk after inoculation is 106～9CFU/mL；
(4) Fermenting the horse milk under the following conditions: fermenting for 58h at 30 ℃ under the conditions of stirring speed of 150rpm and ventilation capacity of 30 vvm;
(5) hydrolyzing with trypsin at 40 deg.C for 0.5 hr after fermentation of horse milk, wherein the mass ratio of protease to fermented horse milk is 5.5%;
(6) three samples of single-spore Saccharomyces cerevisiae fermented milk, trypsin hydrolyzed horse milk product, and horse milk product combining single-spore Saccharomyces cerevisiae fermentation and trypsin hydrolysis were tested for ACE inhibitory activity by HPLC.
The viable count of the yeast in this example is shown in table 1, and the ACE inhibition activities of the three samples in this example are shown in fig. 2, and it is known from the results that the combination of saccharomyces cerevisiae fermentation and trypsin hydrolysis can significantly improve the ACE inhibition rate of the horse milk product, and compared with the saccharomyces cerevisiae fermented milk alone, the ACE inhibition rate is improved by 49.83%, and compared with the horse milk hydrolyzed by trypsin alone, the ACE inhibition rate is improved by 32.03%. Therefore, the ACE inhibition rate of the horse milk product can be obviously improved by combining single-spore saccharomyces cerevisiae fermentation and trypsin hydrolysis;
TABLE 2 viable count of yeasts in horse milk after inoculation
| Sample (I) | Number of viable bacteria |
| Activated bacterial liquid | 6.2×108CFU/mL |
| Horse milk after inoculation | 2.48×107CFU/mL |
Example 3: the production method of the horse milk product of the embodiment is as follows:
(1) inoculating Saccharomyces cerevisiae to YPD liquid culture medium, culturing at 30 deg.C and 220rpm for 13 hr, continuously proliferating for 2 generations, with the inoculum size being 3% by volume, and the viable count of activated bacteria liquid being not less than 108CFU/mL for standby;
(2) centrifuging fresh horse milk, and sterilizing at 115 deg.C for 10 min;
(3) inoculating single-spore Saccharomyces cerevisiae when horse milk is cooled to 30 deg.C, wherein the inoculation amount is 4%, and the viable count in horse milk after inoculation is 106～9CFU/mL；
(4) Fermenting the horse milk under the following conditions: fermenting for 74 hours at the temperature of 30 ℃ and under the conditions of stirring speed of 200rpm and ventilation capacity of 15 vvm;
(5) hydrolyzing with trypsin at 37 deg.C for 1.5 hr after fermentation of horse milk, wherein the mass ratio of protease to fermented horse milk is 6.5%;
(6) three samples of single-spore Saccharomyces cerevisiae fermented milk, trypsin hydrolyzed horse milk product, and horse milk product combining single-spore Saccharomyces cerevisiae fermentation and trypsin hydrolysis were tested for ACE inhibitory activity by HPLC.
The viable count of the yeast in this example is shown in table 3, and the ACE inhibition activities of the three samples in this example are shown in fig. 3, and the results show that the combination of saccharomyces cerevisiae monospora fermentation and trypsin hydrolysis can significantly improve the ACE inhibition rate of the horse milk product, and compared with the saccharomyces cerevisiae fermented milk alone, the ACE inhibition rate is improved by 40.03%, and compared with the horse milk hydrolyzed by trypsin alone, the ACE inhibition rate is improved by 26.23%. Therefore, the ACE inhibition rate of the horse milk product can be obviously improved by combining single-spore saccharomyces cerevisiae fermentation and trypsin hydrolysis.
TABLE 3 viable count of yeasts in horse milk after inoculation
| Sample (I) | Number of viable bacteria |
| Activated bacterial liquid | 3.2×109CFU/mL |
| Horse milk after inoculation | 1.28×108CFU/mL |
Example 4: the production method of the horse milk product of the embodiment is as follows:
(1) inoculating Kluyveromyces marxianus into YPD liquid culture medium, culturing at 28 deg.C and 200rpm for 15 hr, continuously proliferating for 3 generations, with the inoculum size being 1.5% by volume, and the viable count of activated bacteria liquid being not less than 108CFU/mL for standby;
(2) filtering fresh horse milk, and sterilizing at 65 deg.C for 30 min;
(3) inoculating Kluyveromyces marxianus when the temperature of the mare milk is reduced to 28 ℃, wherein the inoculation amount is 2.5%, and the viable count in the inoculated mare milk is 106～9CFU/mL；
(4) Fermenting the horse milk under the following conditions: fermenting for 60h at 28 ℃ under the conditions of stirring speed of 175rpm and ventilation capacity of 10 vvm;
(5) hydrolyzing the fermented horse milk for 4 hours at 50 ℃ by using papain, wherein the mass ratio of the papain to the fermented horse milk is 4.5%;
(6) measuring ACE inhibitory activity of three samples of the separately Kluyveromyces marxianus fermented milk, the separately Kluyveromyces marxianus fermented milk product and the separately Kluyveromyces marxianus fermented milk product combined with the separately Kluyveromyces marxianus fermented milk product and the separately hydrolyzed Kluyveromyces marxianus fermented milk product;
the viable count of the yeast in this example is shown in table 4, and the ACE inhibition activities of the three samples in this example are shown in fig. 4, and the results show that the ACE inhibition rate of the mare milk product can be significantly improved by the combination of kluyveromyces marxianus fermentation and papain hydrolysis, and compared with kluyveromyces marxianus fermented milk alone, the ACE inhibition rate is improved by 41.42%, and compared with the mare milk hydrolyzed by papain alone, the ACE inhibition rate is improved by 30.48%; therefore, the ACE inhibition rate of the horse milk product can be obviously improved by combining the Kluyveromyces marxianus fermentation with the papain hydrolysis;
TABLE 4 viable count of yeasts in horse milk after inoculation
| Sample (I) | Number of viable bacteria |
| Activated bacterial liquid | 5.68×108CFU/mL |
| Horse milk after inoculation | 1.09×107CFU/mL |
Example 5: the production method of the horse milk product of the embodiment is as follows:
(1) inoculating Saccharomyces cerevisiae into YPD liquid culture medium, culturing at 25 deg.C and 250rpm for 14 hr, continuously proliferating for 2 generations, with the inoculum size being 1% by volume, and the viable count of activated bacteria liquid being not less than 108CFU/mL for standby;
(2) centrifuging fresh horse milk, and sterilizing at 95 deg.C for 10 min;
(3) inoculating Saccharomyces cerevisiae when horse milk is cooled to 30 deg.C, wherein the inoculation amount is 4%, and the viable count in horse milk after inoculation is 106～ 9CFU/mL；
(4) Fermenting the horse milk under the following conditions: fermenting for 56h at 30 ℃ under the conditions of stirring speed of 150rpm and ventilation capacity of 14 vvm;
(5) hydrolyzing with flavourzyme at 45 deg.C for 5.5h after horse milk fermentation, wherein the mass ratio of protease to fermented horse milk is 6%;
(6) measuring ACE inhibitory activity of three samples of the horse milk product which is formed by fermenting the independent saccharomyces cerevisiae, hydrolyzing the horse milk product by the flavourzyme, fermenting the saccharomyces cerevisiae and hydrolyzing the flavourzyme by using an HPLC method;
the viable count of the yeast in this example is shown in table 5, and the ACE inhibition activities of the three samples in this example are shown in fig. 5, it can be seen from the results that the combination of saccharomyces cerevisiae fermentation and flavourzyme hydrolysis can significantly improve the ACE inhibition rate of the horse milk product, the ACE inhibition rate is improved by 36.21% compared with that of saccharomyces cerevisiae fermented milk alone, and the ACE inhibition rate is improved by 23.77% compared with that of flavourzyme hydrolyzed horse milk alone, thus the combination of saccharomyces cerevisiae fermentation and flavourzyme hydrolysis can significantly improve the ACE inhibition rate of the horse milk product;
TABLE 5 viable count of yeasts in horse milk after inoculation
| Sample (I) | Number of viable bacteria |
| Activated bacterial liquid | 4.91×108CFU/mL |
| Horse milk after inoculation | 2.03×107CFU/mL |
Claims (8)
1. A method for preparing horse milk products with ACE inhibitory activity by combining yeast fermentation and enzymolysis is characterized in that: fermenting sterilized fresh horse milk with yeast, and performing enzymolysis with protease to obtain horse milk product with ACE inhibitory activity.
2. The method for preparing horse milk product with ACE inhibitory activity according to claim 1, wherein the yeast fermentation is combined with enzymolysis, and the method comprises the following steps: the sterilized fresh horse milk is prepared by filtering or centrifuging fresh horse milk, and sterilizing at 60-150 deg.C for 2 s-30 min.
3. The method for preparing horse milk product with ACE inhibitory activity according to claim 1, wherein the yeast fermentation is combined with enzymolysis, and the method comprises the following steps: the yeast is one or more of single-spore Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris and Dekkera anomala.
4. The method for preparing horse milk product with ACE inhibitory activity according to claim 3, wherein the yeast fermentation is combined with enzymolysis, and the method comprises the following steps: the number of viable bacteria in horse milk after yeast inoculation during fermentation is 106～109CFU/mL。
5. The method for preparing horse milk product with ACE inhibitory activity according to claim 4, wherein the yeast fermentation is combined with enzymolysis, and the method comprises the following steps: the yeast fermented horse milk is fermented for 4-80 hours under the conditions of 20-40 ℃, stirring speed of 5-200 rpm and ventilation volume of 10-600 vvm.
6. The method for preparing horse milk product with ACE inhibitory activity according to claim 1, wherein the yeast fermentation is combined with enzymolysis, and the method comprises the following steps: the protease for enzymolysis is one or more of trypsin, pepsin, neutral protease, alkaline protease, elastase, bromelain, papain, compound protease and flavourzyme, and the mass ratio of the protease to the fermented horse milk is 2-8%.
7. The method of yeast fermentation combined with enzymatic hydrolysis for making horse milk products with ACE inhibitory activity according to claim 6, wherein: hydrolyzing the protease at 28-50 ℃ for 0.5-6 h.
8. Horse milk product obtained by a method for preparing horse milk product with ACE inhibitory activity, which combines yeast fermentation and enzymatic hydrolysis as described in any one of claims 1-7.
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