CN114680213A

CN114680213A - Folum Ilicis fermented tea and its preparation method

Info

Publication number: CN114680213A
Application number: CN202210453133.9A
Authority: CN
Inventors: 周礼红; 谢九三; 解文利; 白雪; 吴雨桐
Original assignee: Guizhou Taihe Modern Ecological Agriculture Technology Co ltd
Current assignee: Guizhou Taihe Modern Ecological Agriculture Technology Co ltd
Priority date: 2022-04-27
Filing date: 2022-04-27
Publication date: 2022-07-01
Anticipated expiration: 2042-04-27
Also published as: CN114680213B

Abstract

The invention discloses a lobular broadleaf holly leaf fermented tea and a preparation method thereof, and relates to the technical field of tea preparation. The preparation method of the lobular broadleaf holly leaf fermented tea comprises the following steps: inoculating yeast to the deactivated folium Ilicis Purpureae, and inoculating the amount of yeast greater than 5 × 10 per gram of the deactivated folium Ilicis Purpureae⁶cfu yeast, which is a patent strain with CCTCC NO of M2014330; inoculating saccharomycetes, and fermenting the ilex latifolia at the temperature of 25-35 ℃ for 10-14 h; and drying after fermentation. The small leaf broadleaf holly leaf fermented tea is prepared by the preparation method provided by the application. The lobular kudingcha fermented tea prepared by the method has clear soup color after being brewed, has pleasant and harmonious fruit fragrance, extremely light bitter and astringent tastes, and obvious aftertaste, and has better taste compared with unfermented lobular kudingcha fermented tea or fermented tea fermented by common yeast.

Description

Folum Ilicis fermented tea and its preparation method

Technical Field

The invention relates to the technical field of tea preparation, in particular to a lobular broadleaf holly leaf fermented tea and a preparation method thereof.

Background

Folum Ilicis is prepared from fresh leaves of Ligustrum quihoui of Ligustrum of Oleaceae, and is also called Folum Ilicis. After the broadleaf holly leaf is refined, the tea soup is fragrant, the lasting fragrance is achieved, and the medicinal value is extremely high.

At present, the lobular broadleaf holly leaves also relate to more patents, such as non-fermented type: CN111838369A A substitute tea formula process of Yuqing lobular Kuding tea; 2020107197868A method for preparing compound Folum Ilicis powder; CN 201911138175.8A method for processing and preparing lobular kudingcha. For example, fermentative type |: 201911067434.2A method for preparing fermented Folum Ilicis.

Aiming at the defects of the prior non-fermented tea: the process is complex, the cost is high, and the problems of introduction of organic solvent and the like exist by adopting some organic solvents for extraction. The fermentation type has the following disadvantages: the mixed fermentation of the Folum Ilicis is carried out by adopting various fungi with large differences in biological characteristics, so that the control of the fermentation process and the judgment of the fermentation end point are difficult to realize, the process is unstable, and the quality of each batch of products is inconsistent.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a lobular broadleaf holly leaf fermented tea and a preparation method thereof.

The invention is realized by the following steps:

in a first aspect, the invention provides a preparation method of a lobular broadleaf holly leaf fermented tea, which comprises the following steps:

inoculating yeast to the deactivated folium Ilicis Purpureae, and inoculating the amount of yeast greater than 5 × 10 per gram of the deactivated folium Ilicis Purpureae⁶cfu yeast, which is a patent strain with CCTCC NO of M2014330;

inoculating saccharomycetes, and fermenting the ilex latifolia at the temperature of 25-35 ℃ for 10-14 h;

and (5) drying after fermentation.

In an optional embodiment, each gram of the ilex microphylla subjected to enzyme deactivation is inoculated with (5-15) multiplied by 10⁶cfu yeast.

Preferably, the yeast is inoculated by inoculating yeast seed solution to the water-removed Folum Ilicis leaves, and the concentration of the seed solution is 1 × 10⁸cfu/ml, the inoculation amount is 0.05-0.15 ml/g.

In an alternative embodiment, before the fermentation of the killed folium Ilicis, a yeast seed solution is prepared, wherein the preparation method comprises:

activating yeast strain to obtain bacterial suspension, and mixing 5 × 10⁸～7×10⁸Inoculating the yeast of cfu into 200mL of culture medium, and carrying out shake culture for 18-20 h under the conditions that the temperature is 23-26 ℃, the initial pH is 6.4-6.5 and the rotation speed of a shaking table is 200-300 rpm.

In an optional embodiment, the drying is performed at 90-120 ℃ until the moisture content is 20-45%, and then at 60-95 ℃ until the moisture content is not more than 10%.

In an optional embodiment, the drying time at 90-120 ℃ is 1-4 min.

In an optional embodiment, the drying time at 60-95 ℃ is 5-12 h.

In an optional embodiment, the method further comprises the step of deactivating enzyme of the fresh folium llicis latifoliae before fermenting the deactivated folium llicis latifoliae, wherein the deactivation of enzyme is as follows: and (5) performing steam de-enzyming for 20-50 min.

In an optional embodiment, after steam enzyme deactivation, cooling to 25-35 ℃ and then carrying out yeast inoculation.

In an alternative embodiment, the Folum Ilicis leaves are picked in the month of 4-9.

In a second aspect, the present invention provides a fermented lobular kudingcha tea, obtainable by a process according to any one of the preceding embodiments.

The invention has the following beneficial effects:

the fermented tea is prepared by adopting a patent strain capable of generating special fragrance after fermentation, and fermenting for 10-14 hours at 25-35 ℃ in an appropriate inoculation amount to effectively neutralize the bitter taste of the lobular ilex, so that the lobular ilex fermented tea which has active substances of the lobular ilex and beneficial substances metabolized by saccharomycetes is obtained.

Compared with the existing method, the method provided by the application has the advantages that the process is simple, the production process can be automatically or semi-automatically carried out, and substances with potential safety hazards such as organic solvents and the like do not need to be introduced; the method only adopts a special yeast for fermentation, the fermentation process is controllable, the process is stable, and the quality of each batch of products is uniform.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a growth curve of yeast;

FIG. 2 is a total ion flow diagram (TIC) in positive ion mode;

FIG. 3 is a total ion flow diagram (TIC) in negative ion mode;

FIG. 4 is a distribution of PC1 for each sample point;

FIG. 5 is a PCA diagram of a quality control sample;

FIG. 6 is a PLS-DA cloud;

FIG. 7 is a graph of the medium PLS-DA metabolite significance;

FIG. 8 is an OPLS-DA dotted cloud;

FIG. 9 is the test statistic for the OPLS-DA displacement test (Q2);

figure 10 is a multiple change volcano plot;

FIG. 11 is a metabolite difference box plot;

FIG. 12 is a statistical graph of the content of the most important 15 metabolites in SVM;

FIG. 13 is an ORA enrichment analysis.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The following is a detailed description of the fermented lobular Kuding tea and the preparation method thereof provided in the examples of the present application.

The preparation method of the lobular broadleaf holly leaf fermented tea provided by the embodiment of the application comprises the following steps:

and (5) drying after fermentation.

According to the method provided by the application, the patent strains capable of generating special fragrance after fermentation are adopted, the bitter and astringent taste of the lobular Kuding can be effectively neutralized after the inoculation of a proper amount and the fermentation for 10-14 h at the temperature of 25-35 ℃, and the lobular Kuding fermented tea which has active substances of the lobular Kuding and beneficial substances metabolized by saccharomycetes is obtained.

Specifically, the preparation method comprises the following steps:

s1, preparing yeast seed liquid

1) Preparation of strains

The strain is as follows: meyerozyma Caribbica Mc186 patent strain, CCTCC NO: M2014330.

2) Preparation of the culture Medium

20.0g of malt extract, 20.0g of glucose and 15.0g of agar, heating and dissolving in a proper amount of water, and fixing the volume to 1000 mL. The pH is natural. Subpackaging and sterilizing at 115 +/-1 ℃ for 20 min.

3) Drawing of growth curves of Yeast strains

The yeast strain is activated to prepare a bacterial suspension, the OD value is 0.50-0.84, the bacterial suspension is inoculated into a 500mL shake flask filled with 200mL culture medium according to the inoculum size of 10% (v/v), and the shake culture is carried out at 30 +/-1 ℃ and 150-. And measuring the OD value of the bacterial liquid every 2h from 0h to 30 h. The growth curve of yeast was plotted with the incubation time as abscissa and the log of the number of colonies as ordinate, as shown in fig. 1.

The growth dynamics curve of the yeast strain shows that the yeast strain enters a stationary phase after 0-2 h, 2-14 h and a logarithmic growth phase after 14 h. Therefore, the culture time is determined to be the early stage of the large stationary phase, and is selected to be 18-20 h.

4) Determination of preparation parameters of Yeast Strain seed solutions

The inventor conducts a great deal of creative work and finds that the inoculation amount is more than 2 multiplied by 10 at the pH of 5-7, the temperature of 25-35 ℃, the rotating speed of 100-300 rpm⁸Seed liquid with considerable viable bacteria can be obtained by culturing cfu under the shaking culture condition. Therefore, the inventors involved orthogonal experiments in the above range to determine the optimal culture conditions.

Selecting L⁹(3⁴) Orthogonal table an orthogonal experiment was performed at a 4-factor 3 level to study yeast strain seed-fluid preparation conditions. The test factors and levels are shown in Table 1, the experimental scheme is shown in Table 2, the experimental results are shown in Table 3, and the influence of the four factors on the results is C from large to small in the fermentation process of the strain>B>A>D, amount of instantly dissolved oxygen>Temperature of>Initial pH>And (5) inoculating the seed.

TABLE 1 Yeast Strain fermentation Quadrature Experimental Table

TABLE 2 orthogonal experimental protocols for fermentation of yeast strains

TABLE 3 Quadrature experimental results for fermentation of yeast strains

As can be seen from the above results, the optimum combination is C₃B₁A₃D₂I.e., when the rotational speed of the shaker is 250rpm, the cultivation temperature is 25 ℃, the initial pH is 6.5, and the inoculation amount is 6X 10⁸The most seed liquid can be obtained or belong to the most seeds when cfu is cultured.

Therefore, in the embodiment of the present invention, the preparation method of the seed solution determined by the above results is:

activating yeast strain to obtain bacterial suspension, and mixing 5 × 10⁸～7×10⁸Inoculating the cfu of yeast into 200mL of culture medium, and carrying out shake culture for 18-20 h at the temperature of 23-26 ℃, the initial pH value and the shaking table rotation speed of 200-300 rpm to obtain yeast seed liquid.

S2, treating Kuding leaf

Picking 4-9 months of small-leaf folium llicis latifoliae leaves or branches with leaves, mechanically taking the leaves and discarding the branches, cleaning the leaves by cleaning equipment, draining, transferring to a steam de-enzyming axe for steam de-enzyming for 20-50 min, transferring to a conveyer belt from the de-enzyming axe, and cooling to about 20-50 ℃ for later use.

S3 inoculation yeast liquid

Inoculating yeast to the deactivated folium Ilicis Purpureae, and inoculating the amount of yeast greater than 5 × 10 per gram of the deactivated folium Ilicis Purpureae⁶The cfu yeast adopts the yeast which is a patent strain with CCTCC NO of M2014330.

Preferably, each gram of the ilex microphylla leaves after fixation is inoculated with (5-15) x 10⁶The cfu yeast can achieve good fermentation effect.

Specifically, the yeast seed liquid is diluted to 1 × 10⁸cfu/ml, then inoculating 0.05-0.15 ml of yeast seed liquid to each gram of ilex latifolia leaves.

S4, fermentation

After inoculation and uniform mixing, the microzyme inoculated Folum Ilicis leaves are conveyed to a fermenter or a fermentation tank, and fermented for 10-14 h (10h, 12h or 14h) at the temperature of 25-35 ℃ (for example, 25 ℃, 30 ℃ or 35 ℃).

Fermenting at the temperature and for a period of time to obtain the lobular Kuding fermented tea which has clear soup color after brewing, pleasant and harmonious fruit flavor and no tartary buckwheat flavor.

S5, drying

And (3) obtaining fermented lobular Kuding leaves after fermentation, transferring the fermented lobular Kuding leaves into a dryer, firstly drying at 90-120 ℃ for 1-4 min until the water content is 20-45%, and then drying at 60-95 ℃ for 5-12 h until the water content is not more than 10% to obtain the lobular Kuding fermented tea.

The ilex latifolia leaves are dried by adopting a gradient temperature drying mode, so that the active ingredients in the ilex latifolia leaves can be effectively prevented from being damaged, and the finally prepared ilex latifolia fermented tea has better taste.

The lobular broadleaf holly leaf fermented tea provided by the embodiment of the application is prepared by adopting the preparation method provided by the embodiment of the application. The fermented tea has clear soup color, pleasant and harmonious fruit flavor, and extremely light bitter and astringent taste, and obvious aftertaste.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

Picking 4-9 months of small-leaf folium llicis latifoliae leaves or branches with leaves, mechanically taking the leaves and discarding the branches, cleaning the leaves by cleaning equipment, draining, transferring to a steam de-enzyming kettle for steam de-enzyming for 20min, transferring to a conveyer belt from the de-enzyming kettle, and cooling to normal temperature for later use.

Inoculating yeast to the deactivated Folum Ilicis leaves, and inoculating to the deactivated Folum Ilicis leaves at a concentration of 1 × 10 per gram⁸cfu/ml yeast seed solution, 0.05ml is inoculated per gram of Folum Ilicis leaves.

After inoculation and uniform mixing, the lobular folium llicis latifoliae inoculated with the microzyme is conveyed into a fermentor or a fermentation tank to be fermented for 12 hours at the temperature of 25 ℃.

And (3) obtaining fermented lobular folium llicis latifoliae after fermentation is finished, transferring the fermented lobular folium llicis latifoliae into a dryer, firstly drying at 100 ℃ for 2min until the water content is about 35%, and then drying at 60 ℃ for 10h until the water content is not more than 10% to obtain the lobular folium llicis latifoliae fermented tea.

Examples 2 to 6

Examples 2-6 are essentially the same as example 1, except that the inoculum size was 0.05ml, 0.07ml, 0.09ml, 0.11ml, 0.13ml and 0.15ml, respectively.

Comparative example 1

This comparative example is essentially the same as example 1 except that: the inoculum size was 0.03 ml.

Comparative examples 2 to 5

Comparative examples 2-5 are essentially the same as example 1, except that the fermentation times were 4, 8, 16 and 20, respectively.

Comparative example 6

This comparative example is essentially the same as example 1, except that fermentation was conducted without inoculating yeast.

Comparative example 7

This comparative example is essentially the same as example 1 except that: common saccharomyces cerevisiae is adopted for fermentation.

Experimental example 1

According to the method embodiments described in examples 1-6 and comparative example 1, the influence of the inoculation amount on the fermentation speed of the Folum Ilicis leaves is examined to measure the viable count while just smelling the fragrance. 3 replicates per experiment. The results are shown in Table 4.

TABLE 4 Effect of different inoculum sizes on fermentation time and viable count

As can be seen from Table 4, the fermentation time was also longer with a lower inoculum size, but there was also a threshold inoculum size at which the fermentation time remained essentially constant. According to the combination of the actual production process and the technical control degree shown in the table 4, the inoculation amount is more than or equal to 5 percent (namely 0.05ml/g) to meet the requirement.

Experimental example 2

3g of the Folum Ilicis fermented tea prepared in example 1 and comparative examples 2-6 are respectively weighed, and soaked in boiling water for 3min, and the tea looks, smells the fragrance and tastes the taste. The results of the identification are reported in table 5.

TABLE 5 sensory differences of Folum Ilicis with different fermentation times

Note: 1. "0" hours means the uninoculated Folum Ilicis matrix; 2. "-" indicates clear and not cloudy as observed by the naked eye.

As can be seen from the table above, the lobular broadleaf holly leaf fermented for 12-20 h is obviously superior to unfermented broadleaf holly leaf in soup color and aroma, and bitter, astringent and sweet aftertaste. However, when the fermentation time is 16 to 20 hours, the broth becomes cloudy and the turbidity increases with the increase in the fermentation time. The experimental results show that the lobular kudingcha leaves with better sensory indexes are products obtained by fermenting yeast for about 12 hours, so that the lobular kudingcha fermented tea with good quality can be obtained when the fermentation time is 10-14 hours.

Experimental example 3

The method for testing the in vitro antioxidant activity of the ilex latifolia leaves before and after fermentation is characterized by comprising the following steps:

1) test method

1. ABTS free radical scavenging test method

2.5ml of ABTS (7mmol/L) were mixed with 44. mu.L of K₂S₂O₈And (140mmol/L) solution is fully and uniformly mixed, and the mixture is kept stand overnight (12 h-16 h) at room temperature under the condition of keeping out of the sun, so as to prepare ABTS + stock solution. The ABTS + stock solution was diluted with PBS buffer (20mmol/L, PH 7.4) and measured with an ultraviolet spectrophotometer so that its absorbance value at an ultraviolet wavelength of 734nm was 0.700+0.002, to prepare an ABTS + working solution. Respectively taking 30uL of each group of samples to be tested, mixing the samples with 3.0ml of ABTS + working solution, fully reacting for 6min, and then measuring the absorbance value at 734nm at normal temperature.

The calculation formula is as follows: clearance rate ═ a_{Blank space}-A_{Sample (I)})/A_{Blank space}×100％

In the formula A_{Blank space}Adding 30uLPBS into 3.0mLABTS + working solution to obtain absorbance value, and detecting vc reference substance with A_{Sample (I)}Is changed to A_{ControlArticle (A)}And (4) finishing.

2. DPPH free radical scavenging experiment

Taking different samples 0.5m L, adding DPPH absolute ethanol solution 1m L with concentration of 0.2mmol/L, mixing uniformly, reacting for 30min at room temperature in a dark place, centrifuging for 10min at 6000 r/min, taking supernatant, measuring absorbance at 517nm, measuring for 3 times in parallel, and taking an average value. The blank group replaced the sample solution with an equal volume of PBS buffer solution with a clearance of

Clearance (%) - (A)_{Blank space}-A_{Sample (I)})/A_{Blank space}×100％。

3. Lipid peroxidation inhibition experiment

Adding PBS into fresh egg yolk at a ratio of 1: 1, magnetically stirring for 10min, and diluting with PBS to obtain egg yolk suspension at a ratio of 1: 25. 1mL of egg yolk suspension, 0.5mL of sample, 1mL of PBS and 1mL of FeSO were taken₄(25mmol/L) of the solution was mixed well and incubated at 37 ℃ for 1.5 h. Then adding 1mL of trichloroacetic acid (TCA, mass fraction is 2.5%), standing at room temperature for 10min, 3500r/min, and centrifuging for 10 min. Taking the supernatant, adding 2mL of thiobarbituric acid (TBA, the mass fraction is 0.8 percent), fully and uniformly mixing, and then carrying out boiling water bath for 10 min; after cooling, the absorbance A was measured at 532nm₁。A₀The expression for the equivalent amount of sample solvent (i.e., PBS) instead of the sample (sun shake, young autumn.) is:

lipid peroxidation inhibition rate ═ a₀-A₁)/A₀×100％。

2) Evaluation results of antioxidant Activity in vitro

TABLE 6 in vitro antioxidant Activity of Folum Ilicis leaves before and after fermentation

Note: comparative example 6, unfermented lobular Kuding leaf; example 1 fermented Folum Ilicis leaves

As can be seen from Table 6, the evaluation test of the in vitro antioxidant model shows that the ABTS clearance and DPPH clearance before and after the fermentation of the Folum Ilicis leaves are obviously different (p is less than or equal to 0.05), but the actual change is not too large. In addition, the lipid peroxidation resisting activity of the Folum Ilicis leaves after fermentation is obviously improved (p is less than or equal to 0.01) compared with that before fermentation, and is 1.93 times that before fermentation (see Table 6).

Experimental example 4

The difference between the material composition and distribution of the fermented lobular folium Ilicis and unfermented lobular folium Ilicis is explored.

The fermented (example 1) and unfermented (comparative example 6) lobular Kuding tea were compared analytically using non-targeted metabolome techniques and analytical methods to see the differences in material composition and distribution between the two.

1) Mass spectrometric data analysis results

Based on a high performance liquid chromatography-high resolution mass spectrometry (UHPLC-QE-MS) technology, Agilent mass spectrometry b.08.00software (Agilent Technologies, USA) is adopted to perform universal format conversion on raw data, an XCMS program package is used to perform preprocessing such as peak identification and internal standard normalization, and a visualization matrix (such as fig. 2 and fig. 3) of a peak area is obtained, fig. 2 is a total ion flow graph (TIC) in a positive ion mode, and fig. 3 is a total ion flow graph (TIC) in a fluorine ion mode. The results of fig. 2 and 3 show that the experimental method is stable and reliable and has good repeatability.

2) Data Quality Assessment (QA) and Quality Control (QC)

The distribution of the PC1 values of all the sample points is used to evaluate whether the laboratory sample preparation and sample measurement process is controllable, as shown in fig. 4, fig. 4 shows the distribution of PC1 of each sample point, the abscissa shows the detection sample, and the ordinate shows the PC1 value of the sample in the PCA analysis. The results of fig. 4 show that the data has controllability.

And (3) evaluating the signal drift and the correction quality of the whole mass spectrum data in the acquisition process by using a quality control sample (QC sample), wherein as shown in the result of fig. 5, fig. 5 is a PCA (principal component analysis) diagram of the quality control sample, red points are corrected quality control sample points (QC samples), and blue points are detection samples. QC sample points were clustered together, indicating good calibration.

3) Partial least squares discriminant analysis (PLS-DA) analysis

The samples were analyzed using a supervised multivariate statistical method, partial least squares discriminant analysis (PLS-DA), and the results are shown in FIGS. 6 and 7.

FIG. 6 is a PLS-DA cloud plot, where GO3 was the unfermented group; GO5 is a fermentation group. Each point in the graph corresponds to a sample, and the horizontal and vertical coordinates are the values of the two factors with the best discrimination effect. The different groupings are marked with different colors and the area marked by an ellipse is the 95% confidence region for the sample point. As can be seen from FIG. 6, the sample points of different groups are distributed in different areas, which indicates that the PLS-DA model has better discrimination effect and metabolites with significant differences exist among the groups.

FIG. 7 is a PLS-DA metabolite significance plot, where each point represents a metabolite, VIP (value Import in project) on the abscissa and p-value after FDR correction (log10 transformation) on the ordinate. The most valuable metabolites (VIP > 1) that contributed significantly to the discriminant analysis were found in FIG. 7, and these metabolites could be used as biomarker (biomaker) to distinguish different groups. The metabolites marked in the yellow region, i.e., those with corrected p-value less than 0.05 and VIP greater than 1, were significantly different between groups, indicating that there was a significant difference in the composition and distribution of the material between the fermented and unfermented groups.

4) Quadrature partial least squares discriminant analysis

Since the PLSDA analysis can show overfitting, an Orthogonal Partial Least Squares Discriminant Analysis (OPLSDA) was used for the improvement. As shown in fig. 8 and 9, fig. 8 is an OPLS-DA dotted cloud in which GO3 is the unfermented group; GO5 is a fermentation group. In the figure, each point corresponds to one sample, and the horizontal and vertical coordinates are values of two factors with the best discrimination effect. The different groupings are marked with different colors and the area marked by the ellipse is the 95% confidence area of the sample point. FIG. 9 shows the distribution of test statistics (Q2) and p-values for the OPLS-DA displacement test, the distribution being the random distribution of displacements of Q2, the arrows referring to the actual observed model Q2.

Through OPLSDA analysis, sample points of different groups are distributed and gathered in different areas, and through replacement inspection, Q2 is actually observed on the right side of random distribution, and p is less than 0.01, which shows that the discrimination effect of the OPLS-DA model is good, and metabolites with obvious difference exist between a fermentation group and an unfermented group.

5) Univariate analysis (05-Univariate analysis catalog)

Significant differential changes in metabolites between the fermented and unfermented groups have been determined by analysis of PLSDA, OPLSDA, but the magnitude of this change is not given. To measure and evaluate how large this Change is, we screened metabolites with significant changes using a method of calculating Fold Change (FC) of metabolite changes in combination with p values. And in default, the group with the group name arranged in the front according to the alphanumeric sequence is used as a reference, the change multiple of the mean value of the other group and the mean value of the reference group is calculated, the up-modulation multiple is positive, and the down-modulation multiple is negative. As in fig. 10, fig. 10 is a multiple change volcano plot, each point represents a metabolite, the abscissa is the multiple of change, the ordinate is the T-test p-value, the greater the multiple of change, the smaller the p-value (the higher log10 (p)), the larger the point. As can be seen from fig. 10, the metabolites in the yellow region are those with p less than 0.05 and with fold absolute values greater than 2, with significant differences between the fermented and unfermented cohorts. In addition, to visually demonstrate the differences in metabolites between groups, a box plot of representative differential metabolites ranked top (top 25 with smaller P-value) obtained from one-dimensional statistical analysis was made (see fig. 11). In fig. 11, p <0.05, p <0.01, and p <0.001, respectively.

6) Support vector machine screening of inter-group important differential metabolites

A bagging method is combined with a Support Vector Machine (SVM), namely the SVM is trained by using the k training sets of Bootstraping and independently selecting the most important metabolites in each training set. The frequency of each metabolite selected in k training sets is calculated and used to measure the importance of the metabolite in the SVM analysis, and the average importance of the metabolite in all training sets is also calculated. Through calculation, 26 metabolites with significant differences existing between the fermentation group and the non-fermentation group are screened out, and are metabolites which are up-regulated in the fermentation group, wherein the most important 15 metabolites are shown in figure 12, and in figure 12, GO3 is shown in the non-fermentation group; GO5 is a fermentation group. The abscissa of the left panel is the mean importance of SVM and the right panel is a heat map of the content of metabolites in two groups in 15. Wherein, the palmitoleic acid, the inositol, the tryptophan, the pantothenic acid, the D-xylose and other substances which are obviously up-regulated in a fermentation group have better physiological activity. These important substances can be used as candidate markers for distinguishing the fermented group from the unfermented group.

7) Metabolic pathway analysis

Through metabolic pathway enrichment analysis, the substance metabolic pathways which are remarkably changed between fermentation groups and non-fermentation groups are mainly related to the metabolic pathways of amino acids, purines, fatty acids, vitamins, TCA, fructose, mannose, pentose, caffeine, pyridine, pyrrole, ubiquinone and other terpenoid quinones, and the like, and is shown in FIG. 13, wherein GO3 is the non-fermentation group; GO5 is a fermentation group. The abscissa is the fold enrichment, which is the observed/theoretical metabolite number in the metabolic pathway. The magnitude of the p-value is expressed in color, with darker colors yielding smaller p-values.

Through the analysis, the variety and the content of active ingredients in the lobular broadleaf holly leaf can be obviously improved after the lobular broadleaf holly leaf is fermented by the method provided by the application.

Experimental example 5

Whether the yeast (example 1) and the common saccharomyces cerevisiae (comparative example 7) related to the application are suitable for fermenting the ilex microphylla leaves is examined, and fermentation tests are carried out by using the same pretreatment method of the ilex microphylla leaves and the same seed solution concentration, inoculation amount, fermentation temperature and fermentation time by taking fragrance, alcohol taste, liquor color and taste as indexes. The results show that the leaves of the ilex microphylla are fermented by the aroma-producing yeast, and the leaves have better performance in terms of liquor color, aroma and taste, while the saccharomyces cerevisiae can produce strong alcoholic smell with vinasse taste, turbid liquor color and does not meet the basic requirements of sense. Therefore, the patent aroma-producing yeast is selected as the fermentation strain of the small leaf Kuding leaf.

TABLE 7 sensory evaluation of the bitter lobular products of fermentation of different yeast strains

As can be seen from Table 7, the obtained broadleaf holly leaf tea by fermenting with the specific aroma-producing yeast is clearer in liquor color, better in aroma, free of alcohol taste and bitter taste and obviously better in comprehensive quality compared with the common saccharomyces cerevisiae.

In conclusion, the preparation method of the broadleaf holly leaf fermented tea provided by the application adopts the patent bacterial strain which can generate special fragrance after fermentation, the bitter and astringent taste of the broadleaf holly leaf can be effectively neutralized by inoculating a proper amount of the patent bacterial strain and fermenting the patent bacterial strain at the temperature of 25-35 ℃ for 10-14 h, and the broadleaf holly leaf fermented tea which has active substances of the broadleaf holly leaf and beneficial substances metabolized by saccharomycetes is obtained. More importantly, compared with the existing method, the method provided by the application has the advantages that the process is simple, the production process can be automatically or semi-automatically carried out, and substances with potential safety hazards such as organic solvents and the like do not need to be introduced; according to the method, only one special yeast is adopted for fermentation, the fermentation process is controllable, the process is stable, and the quality of each batch of products is uniform.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A preparation method of lobular broadleaf holly leaf fermented tea is characterized by comprising the following steps:

inoculating yeast to the deactivated folium Ilicis Purpureae, and inoculating the yeast to the deactivated folium Ilicis Purpureae at a ratio of 5 × 10/g⁶cfu yeast, which is a patent strain with CCTCC NO of M2014330;

and (5) drying after fermentation.

2. The method for producing the fermented lobular Kuding tea according to claim 1, wherein the method comprisesCharacterized in that (5-15) multiplied by 10 is inoculated on each gram of the water-removed lobular Kuding leaves⁶cfu yeast;

preferably, the yeast is inoculated by inoculating yeast seed solution to the water-removed lobular ilex latifolia leaves, and the concentration of the seed solution is 1 x 10⁸cfu/ml, and the inoculation amount is 0.05-0.15 ml/g.

3. The method of claim 2, further comprising preparing the yeast seed solution prior to fermenting the de-enzymed Folum Ilicis leaves, the method comprising:

4. The method for preparing fermented lobular kudingcha according to claim 1, wherein the drying is performed by drying at 90-120 ℃ until the moisture content is 20-45%, and then drying at 60-95 ℃ until the moisture content is not more than 10%.

5. The method for preparing the Folum Ilicis fermented tea according to claim 4, wherein the drying time at 90-120 ℃ is 1-4 min.

6. The method for preparing the Folum Ilicis fermented tea according to claim 4, wherein the drying time at 60-95 ℃ is 5-12 h.

7. The method for preparing a fermented lobular Kuding tea according to claim 1, wherein the method further comprises deactivating enzymes of fresh lobular Kuding leaves before fermenting the deactivated lobular Kuding leaves, wherein the deactivating enzymes are as follows: and (5) performing steam de-enzyming for 20-50 min.

8. The method for preparing the lobular Kuding fermented tea according to claim 7, wherein the inoculation of yeast is performed after the steam de-enzyming and cooling to 25-35 ℃.

9. The method for preparing the Folum Ilicis fermented tea according to claim 1, wherein the Folum Ilicis leaves are picked in 4-9 months.

10. A fermented lobular Kuding tea, characterized in that it is prepared by the process according to any one of claims 1 to 9.