CN109123647B - Preparation method of black barley enzyme capable of effectively enriching gamma-aminobutyric acid and polyphenol - Google Patents

Preparation method of black barley enzyme capable of effectively enriching gamma-aminobutyric acid and polyphenol Download PDF

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CN109123647B
CN109123647B CN201811220405.0A CN201811220405A CN109123647B CN 109123647 B CN109123647 B CN 109123647B CN 201811220405 A CN201811220405 A CN 201811220405A CN 109123647 B CN109123647 B CN 109123647B
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宋立华
蒋芮
管玘
丁信文
欧阳鹏凌
宋晨玮
吴佳怡
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Abstract

The invention discloses a preparation method of black barley enzyme for effectively enriching gamma-aminobutyric acid and polyphenol, which comprises the following steps: 1) preparing a black barley enzymolysis liquid culture medium: comprises the steps of cleaning, crushing, enzymolysis, sterilization and cooling of the black barley to obtain the black barley enzymolysis liquid culture medium; 2) activating the strain of lactobacillus producing gamma-aminobutyric acid by using a culture medium to obtain a seed solution; 3) inoculating the seed liquid obtained in the step 2) into the black barley enzymolysis liquid culture medium obtained in the step 1), and fermenting to obtain black barley fermentation liquid. The invention screens lactobacillus which can be used for fermenting the black barley and effectively enrich gamma-aminobutyric acid and polyphenol, and establishes a culture medium preparation process for processing the black barley enzyme. The invention can provide a new choice for further developing the functional cereal ferment food taking the black barley as the main raw material, thereby having better application prospect.

Description

Preparation method of black barley enzyme capable of effectively enriching gamma-aminobutyric acid and polyphenol
Technical Field
The invention relates to the technical field of grain and functional food processing, in particular to a preparation method of black barley enzyme capable of effectively enriching gamma-aminobutyric acid and polyphenol.
Background
Whole grains are grains that are dehulled and then composed of a cortex, germ, and endosperm. The cortex contains a large amount of dietary fiber and mineral substances, and the embryo contains a large amount of nutrient substances, so that the whole grain has richer nutrient components. Research has shown that the whole grain food can effectively reduce the risk of cardiovascular and cerebrovascular diseases, diabetes, malignant tumor and other related chronic diseases. However, because the whole grain cortex contains a large amount of cellulose, the whole grain cortex is directly eaten as staple food and is not beneficial to normal digestion and absorption of a human body; in addition, the whole grain food has poor water absorption and expansibility, long cooking time and poor taste, so the whole grain food is not easy to be accepted by people. The whole grain ferment obtained by fermentation can not only fully utilize the original nutrient components, improve some original bad flavors of the fermentation substrate, but also can generate some new functional components after fermentation, thereby further increasing the nutrient value of the whole grain.
The whole grain ferment is a nutrient mixture obtained by adding a small amount of honey into whole grains as a substrate and fermenting by using probiotics such as active baker's yeast, lactobacillus and the like. At present, the research on the cereal enzymes is few in products on the market at home, and the products are different in quality, so that the industrial production is not formed. Although the enzyme products such as the high-brown rice enzyme have been produced in advance in this respect in japan, the "black barley enzyme" product using black barley as a fermentation substrate and the invention thereof have not been reported yet.
The existing research results show that the grain ferment fermentation process conditions are different, and the types and the contents of the gamma-aminobutyric acid and other functional active substances are greatly different, so that how to obtain the whole grain ferment food with higher active ingredients such as gamma-aminobutyric acid (GABA), polyphenol and the like through the optimization of a culture medium, the screening of strains and raw materials and the optimization of the fermentation process is a problem worthy of deep research.
Therefore, those skilled in the art have made an effort to develop a method for preparing black barley enzyme that is effectively enriched in γ -aminobutyric acid and polyphenols.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention provides a method for efficiently enriching active ingredients such as γ -aminobutyric acid and polyphenols from black barley.
The invention discloses a preparation method of black barley enzyme for effectively enriching gamma-aminobutyric acid and polyphenol, which comprises the following steps:
1) preparing a black barley enzymolysis liquid culture medium: comprises the steps of cleaning, crushing, enzymolysis, sterilization and cooling of the black barley to obtain the black barley enzymolysis liquid culture medium;
2) activating the strain of lactobacillus producing gamma-aminobutyric acid by using a culture medium to obtain a seed solution; the Lactobacillus producing gamma-aminobutyric acid is Lactobacillus hilgardii (Lactobacillus hilgardii) DSMZ 20051, Lactobacillus newcastle (Lactobacillus diolivorans) DSMZ 14421 and Lactobacillus kisonensis (Lactobacillus kisonensis) JCM15041 which are separated in 2002;
3) inoculating the seed liquid obtained in the step 2) into the black barley enzymolysis liquid culture medium obtained in the step 1), and fermenting to obtain black barley fermentation liquid.
Among them, Lactobacillus Kisonensis (Lactobacillus Kisonensis) is named by the name of Kisonensis, the finder thereof.
Further, the black barley is cleaned, crushed, sieved by a 40-mesh sieve and then subjected to enzymolysis.
Further, the enzymolysis conditions are as follows: the mass ratio of the black barley to the water, namely the feed-liquid ratio is 1:5-10, preferably 1: 5; the enzyme used for enzymolysis is alpha-high temperature amylase, and further, the addition amount of the enzyme is 10U/g; the enzymolysis temperature is 65-75 ℃, and the preferable temperature is 70 ℃; the enzymolysis time is 20-60min, preferably 40 min.
Further, the enzymolysis termination conditions are as follows: after the enzymolysis is finished, the temperature is quickly raised to 100 ℃, and the mixture is boiled for 15 min.
Further, the enzymolysis liquid is sterilized after the enzymolysis is finished.
Preferably, the sterilization conditions of the enzymolysis liquid are as follows: cooling at 115 deg.C for 15min to obtain black barley enzymolysis liquid culture medium.
Further, the strain of lactobacillus producing gamma-aminobutyric acid is activated by MRS culture medium to obtain seed liquid.
Further, the activated lactobacillus seed liquid for producing the gamma-aminobutyric acid is inoculated to a black barley enzymolysis liquid culture medium, wherein the inoculation amount is 3-9% by volume percent, and is preferably 3-5%.
Further, the fermentation time is 24-72h, preferably 48 h.
Preferably, Lactobacillus kisonensis (Lactobacillus kisonensis) JCM15041 is inoculated into the black barley enzymolysis liquid medium.
Preferably, the inoculation amount is 3 percent by volume, the fermentation temperature is 30 ℃, and the fermentation time is 24 hours.
In some embodiments, step 1) is specifically: cleaning and crushing black barley, sieving the black barley with a 40-mesh sieve, adding pure water, heating and stirring the materials according to the mass ratio of 1:5 when pasting, adding alpha-high-temperature amylase according to the addition amount of 10U/g when the enzymolysis temperature reaches 70 ℃, carrying out enzymolysis for 40 minutes, then quickly heating to 100 ℃, boiling for 15 minutes, sterilizing at 115 ℃ for 15 minutes, and cooling to obtain a black barley enzymolysis liquid culture medium; the step 3) is specifically as follows: inoculating Lactobacillus kisonensis (Lactobacillus kisonensis) JCM15041 into the black barley zymolysis solution culture medium obtained in the step 1), wherein the inoculation amount is 3%, and fermenting for 24 hours at 30 ℃.
Furthermore, the screened lactobacillus is not only suitable for the black barley, but also can be widely applied to the fermentation process of brown rice, quinoa, beans and other foods.
Further, the preparation method of the black barley enzyme can obviously increase the content of other functional components such as essential fatty acid gamma-linoleic acid and the like, and adds procyanidine B2And the like.
The invention firstly screens lactobacillus which can be used for fermenting the black barley and effectively enrich gamma-aminobutyric acid and polyphenol, and establishes a culture medium preparation process and an optimal fermentation process condition for processing the black barley enzyme. Under the condition of the fermentation process, the active ingredients such as gamma-linoleic acid, malic acid and epicatechin are obviously increased, and catechin, coumaric acid and procyanidine B are added2And the like. The invention can provide a new choice for further developing the functional cereal ferment food taking the black barley as the main raw material, and has better application prospect.
The conception, the specific steps, and the technical effects produced by the present invention will be further described in conjunction with the accompanying drawings to fully understand the objects, the features, and the effects of the present invention.
Drawings
FIG. 1 is a process flow of ferment fermentation of black barley according to a preferred embodiment of the present invention;
FIG. 2 shows the result of qualitative detection of gamma-aminobutyric acid by paper chromatography according to a preferred embodiment of the present invention;
FIG. 3 shows the result of quantitative determination of gamma-aminobutyric acid by the amino acid analyzer according to the preferred embodiment of the present invention;
FIG. 4 shows active ingredients in the fermented product of black barley according to a preferred embodiment of the present invention in significantly varying amounts;
FIG. 5 shows the types and proportions of the newly added active ingredients in the fermented product of black barley according to a preferred embodiment of the present invention.
Detailed Description
The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.
Example 1
The present example is the preparation of a black barley enzymolysis liquid culture medium:
as shown in table 1, the influence of the gelatinized material-liquid ratio, the enzymolysis temperature and the enzymolysis time on the content of reducing sugar in the black barley zymolyte is examined by adopting a three-factor three-level orthogonal test, and the preparation process conditions for optimally establishing the black barley zymolyte culture medium are as follows: crushing black barley, sieving the crushed black barley with a 40-mesh sieve, and sequentially mixing the raw materials according to a material-liquid ratio of 1:5(m/m) adding pure water, heating, raising the temperature and stirring; when the temperature reaches 70 deg.C, adding alpha-high temperature amylase (10U/g, purchased from Shanghai Aladdin Biotechnology, Ltd.) for 40 min; after the enzymolysis time is over, quickly heating to 100 ℃, and boiling for 15 min; finally, sterilizing at 115 ℃ for 15min, and cooling for later use. The influence of 70 ℃ and 75 ℃ on the enzymolysis result is not very different, and the enzymolysis temperature can be 70 ℃ from the cost-saving perspective.
TABLE 1 factor orthogonal test L for affecting reducing sugar content of black barley zymolyte9(34) Results
Figure BDA0001834616810000031
Figure BDA0001834616810000041
Example 2
This example is a screening of a lactobacillus producing gamma-aminobutyric acid:
the black barley enzymolysis liquid is used as a culture medium to establish a black barley fermentation process flow, as shown in figure 1. Activating the strain with MRS culture medium to form seed liquid, and inoculating to cooled black barley enzymolysis liquid culture medium for fermentation to obtain black barley fermentation liquid. And using gamma-aminobutyric acid as an index, using the black barley enzymolysis liquid as a culture medium, and respectively inoculating 21 lactobacillus strains in the black barley culture medium, wherein 11 lactobacillus plantarum strains (the numbers are respectively 11, 35, 25, 294, 2519, 34, 3736, 16, 234, 30 and 01) are separated and stored from food microbiology laboratory cornel of Shanghai traffic university (wherein the number 34 is lactobacillus casei, and the rest are lactobacillus plantarum); the origin and provenance of the other 10 strains are shown in Table 2.
TABLE 2 sources and sources of part of the fermentation tubes used in the present invention
Figure BDA0001834616810000042
Figure BDA0001834616810000051
Note: number 1 in this table is not the same strain as number 01 isolated and stored from cornel, a food microbiology laboratory of shanghai university of transportation.
The experimental results show that the bacteria No. 7, 9 and 10 have certain capacity of enriching the gamma-aminobutyric acid, as shown in the figure 2 and the figure 3. The "+" in fig. 3 indicates significant difference compared to the blank (unfermented black barley zymolyte broth), P < 0.05. On the basis, through orthogonal experiments, the fermentation process conditions of the black barley capable of effectively enriching the gamma-aminobutyric acid are optimized and established. 1%, 3% and 5% of activated bacteria No. 7, 9 and 10 are respectively inoculated in a black barley zymolyte liquid culture medium, the samples are sampled and tested for gamma-aminobutyric acid after the culture is carried out for 24 hours under the conditions of initial pH 6.5 and 30 ℃, and the results of orthogonal experiments are shown in Table 3. The orthogonal experiment result shows that: the optimal fermentation condition is strain No. 10, the fermentation time is 24 hours, and the inoculation amount is 5%. Under the condition, the enrichment amount of the gamma-aminobutyric acid is 43.56mg/100g, and is improved by 1.34 times compared with an unfermented control group (18.59mg/100 g).
TABLE 3 orthogonal test results for fermentation enrichment of gamma-aminobutyric acid using black barley liquid medium
Figure BDA0001834616810000052
Figure BDA0001834616810000061
Example 3
The embodiment is the establishment and optimization of technological parameters for simultaneously enriching polyphenol and gamma-aminobutyric acid in black barley fermentation:
the optimized fermentation process orthogonal experiment is established by taking polyphenol as a research index, and the experimental result is shown in table 4. The result shows that the technological parameters for optimally enriching the polyphenol in the black barley fermentation product are as follows: bacterium No. 10, inoculum size 9%, feed liquid ratio 1: fermenting at 30 deg.C for 24 h.
TABLE 4 orthogonal experimental results of polyphenol enrichment by fermentation of black barley liquid medium
Figure BDA0001834616810000062
Figure BDA0001834616810000071
Considering comprehensively that both the gamma-aminobutyric acid and the experimental result are better than that of the bacterium No. 10, the enrichment conditions of the polyphenol and the gamma-aminobutyric acid of the bacterium No. 10 under the conditions of the inoculation amounts of 3%, 6% and 9% are further verified, and the results are shown in tables 5 and 6, wherein the enrichment effect of the 3% inoculation amount is higher than that of the 6% and 9%, and the enrichment effect of the 3% inoculation amount is as follows: the polyphenol content is 88mg/100g, which is improved by 1.05 times compared with the unfermented group (43mg/100 g); the gamma-aminobutyric acid also reaches the maximum 65.14mg/100g, which is improved by 2.42 times compared with the unfermented group (19.06mg/100 g). In consideration of the actual production cost, 10 bacteria was used, and the inoculum size was 3%.
TABLE 5 results of verifying polyphenol content in fermented product of black barley under optimum conditions (fungus No. 10)
Figure BDA0001834616810000072
P <0.0001vs unfermented control group
TABLE 6 content of gamma-aminobutyric acid (mg/100g) under optimal fermentation process conditions for polyphenol production
Figure BDA0001834616810000073
In conclusion, the process conditions for effectively enriching the active ingredients such as the gamma-aminobutyric acid, the polyphenol and the like by taking the black barley enzymolysis liquid as the culture medium and the lactobacillus as the fermentation strain are as follows: crushing black barley, sieving the black barley with a 40-mesh sieve, sequentially adding pure water according to a designed proportion, heating and stirring the mixture according to a ratio of 1:5(m/m) of material liquid during gelatinization, adding alpha-high-temperature amylase (with the addition amount of 10U/g) when the mixture reaches an enzymolysis temperature of 70 ℃, carrying out enzymolysis for 40min, then quickly heating the mixture to 100 ℃, boiling the mixture for 15min, sterilizing the mixture at 115 ℃ for 15min, cooling the mixture, inoculating No. 10 bacteria, wherein the inoculation amount is 3%, and the fermentation time is 24h, under the condition, the content of gamma-aminobutyric acid is 65.14mg/100g, which is 2.42 times higher than that of an unfermented control group (19.06mg/100g), the polyphenol content is 88mg/100g, and is 1.05 times higher than that of a control group (43mg/100 g).
In addition to the measurement of polyphenol content, UPLC-MS analysis of black barley fermented product showed that the content of other active ingredients changed after fermentation, and fig. 4 shows the portion of active ingredients with a large content change after fermentation. It can be seen that the functional active components such as essential fatty acid gamma-linoleic acid, malic acid, hesperetin and epicatechin are remarkably increased after fermentation.
In addition to the above active ingredients, UPLC-MS analysis also found that new active substances not contained in the grain before fermentation could be produced after the black barley was fermented, as shown in fig. 5. Mainly comprises catechin, salvianolic acid, coumaric acid, and procyanidin B2And the like.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims (5)

1. A preparation method of black barley enzyme capable of effectively enriching gamma-aminobutyric acid and polyphenol is characterized by comprising the following steps:
1) preparing a black barley enzymolysis liquid culture medium: comprises the steps of cleaning, crushing, enzymolysis, sterilization and cooling of the black barley to obtain the black barley enzymolysis liquid culture medium;
2) activating the strain of lactobacillus producing gamma-aminobutyric acid by using a culture medium to obtain a seed solution; the Lactobacillus producing gamma-aminobutyric acid is Lactobacillus hilgardii (Lactobacillus hilgardii) DSMZ 20051, Lactobacillus newcastle (Lactobacillus diolivorans) DSMZ 14421 or Lactobacillus kisonensis (Lactobacillus kisonensis) JCM15041 which is separated in 2002;
3) inoculating the seed liquid obtained in the step 2) into the black barley enzymolysis liquid culture medium obtained in the step 1), and fermenting to obtain black barley fermentation liquid;
wherein the enzymolysis condition in the step 1) is the mass ratio of the black barley to the water, namely the material-liquid ratio is 1: 5-10; the enzyme used in the enzymolysis in the step 1) is alpha-high temperature amylase; the enzymolysis temperature in the step 1) is 65-75 ℃; the enzymolysis time is 20-60 min; the inoculation amount in the step 3) is 3-9% by volume percentage; the fermentation time in the step 3) is 24-72 h.
2. The method for preparing the black barley enzyme according to claim 1, wherein the black barley enzyme is subjected to enzymolysis after being crushed in the step 1) and passing through a 40-mesh sieve.
3. The black barley enzyme preparation method according to claim 1, wherein the culture medium in the step 2) is an MRS medium.
4. The method for preparing black barley enzyme according to claim 1, wherein the step 1) is specifically: cleaning and crushing black barley, sieving the black barley with a 40-mesh sieve, adding pure water, heating and stirring the materials according to the mass ratio of 1:5 when pasting, adding alpha-high-temperature amylase according to the addition amount of 10U/g when the enzymolysis temperature reaches 70 ℃, carrying out enzymolysis for 40min, then quickly heating to 100 ℃, boiling for 15min, sterilizing at 115 ℃ for 15min, and cooling to obtain the black barley enzymolysis liquid culture medium.
5. The method for preparing black barley enzyme according to claim 1, wherein the step 3) comprises: inoculating Lactobacillus kisonensis (Lactobacillus kisonensis) JCM15041 into the black barley zymolysis solution culture medium obtained in the step 1), wherein the inoculation amount is 3%, and fermenting for 24h at 30 ℃.
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CN105942084A (en) * 2016-04-28 2016-09-21 天津科技大学 Method for producing probiotic functional food through buckwheat fermentation

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