阅读说明：本技术 一种复合菌种及其制备植物酵素的方法 (Composite strain and method for preparing plant enzyme by using same ) 是由 陈艳红 倪辉 杨远帆 李利君 黄君阳 于 2021-05-19 设计创作，主要内容包括：本发明公开了一种复合菌种及其制备植物酵素的方法,该复合菌种包括戊糖片球菌和植物乳杆菌。该复合菌种不仅可以提高发酵产物中的活性物质,例如葡萄白藜芦醇和总酚含量,还能降低传统植物产品经过植物乳杆菌发酵后产生的异味及酸味,并赋予植物酵素产品怡悦的果香与甜香,从而大幅度改良产品的风味。(The invention discloses a composite strain and a method for preparing plant enzyme by using the same. The compound strain can not only improve the content of active substances in a fermentation product, such as resveratrol and total phenols of grapes, but also reduce the peculiar smell and sour taste of a traditional plant product after lactobacillus plantarum fermentation, and endow the plant enzyme product with pleasant fruit aroma and sweet aroma, thereby greatly improving the flavor of the product.)

1. The composite strain for preparing the plant enzyme is characterized by comprising pediococcus pentosaceus and lactobacillus plantarum.

2. The complex strain for preparing plant ferment of claim 1, wherein the lactobacillus plantarum is ATCC8014 and the pediococcus pentosaceus is ATCC 33316.

3. The complex strain for preparing plant ferment as claimed in claim 1, wherein the ratio of the pediococcus pentosaceus to the lactobacillus plantarum is 1: 1.

4. A method for preparing plant ferment is characterized by comprising the following steps:

(1) mixing pediococcus pentosaceus and lactobacillus plantarum to obtain a composite strain;

(2) cleaning plant raw materials, adding water, adjusting the pH value to 3.5-4.0, sterilizing at 60 ℃ for 30min, cooling to room temperature, inoculating the composite strain, and fermenting to obtain the plant enzyme.

5. The method according to claim 4, wherein in the step (1), Pediococcus pentosaceus and Lactobacillus plantarum are activated and grown to logarithmic growth phase, respectively, and mixed after counting; mixing to obtain a mixture with a total bacteria concentration of 5 × 10 or more⁶The ratio of the number of the pediococcus pentosaceus to the CFU/mL bacterial liquid is 20-80%.

6. The method according to claim 4, wherein in the step (2), the plant material is grape, grapefruit or edible seaweed.

7. The method according to claim 4, wherein in the step (2), the fermentation temperature is 20 ℃ to 30 ℃ and the fermentation time is 3 to 15 days.

8. The method of claim 4, wherein in step (2), the initial number of the composite bacterial species is 5 x 10 or more⁴CFU/mL。

Technical Field

The invention relates to the technical field of food fermentation, in particular to a composite strain and a method for preparing plant enzyme by using the same.

Background

In recent years, plant enzyme food has attracted more and more attention in the field of functional food industry, namely the heterophoria, and the promotion effect on human health of the plant enzyme food is attracting more and more attention.

In the production process of modern plant enzyme, artificial inoculation of lactic acid bacteria is often adopted for fermentation production. Some lactic acid bacteria, such as lactobacillus plantarum, have been widely used in the production of plant ferment products, and the content of active ingredients is significantly increased after fermentation. For example, the resveratrol content of grapes is obviously improved after the fermentation of lactobacillus plantarum, and the grapes have better inoxidizability and the efficacy of preventing cancers and cardiovascular diseases. However, the plant ferment products in the related art often have severe sour and musty taste, and the loss of fruit and sweet flavors is severe, which affects the flavor of the ferment products.

Disclosure of Invention

In order to solve the above problems, the present invention provides a composite strain and a method for preparing a plant ferment, wherein the strain can simultaneously improve the biological activity and flavor quality of the plant ferment.

In order to achieve the above objects, embodiments of the present invention in one aspect provide a complex strain for preparing plant enzymes, which includes pediococcus pentosaceus and lactobacillus plantarum.

According to the composite strain for preparing the plant ferment, disclosed by the embodiment of the invention, the content of active substances (such as resveratrol and total phenols) in a fermentation product can be increased, the peculiar smell and sour taste generated after the traditional plant product is fermented by lactobacillus plantarum can be reduced, and pleasant fruit fragrance and sweet fragrance are given to the plant ferment product, so that the flavor of the product is greatly improved.

Optionally, the lactobacillus plantarum is ATCC8014 and the pediococcus pentosaceus is ATCC 33316.

Optionally, the ratio of pediococcus pentosaceus to lactobacillus plantarum is 1: 1.

In another aspect, the present invention provides a method for preparing plant ferment, comprising the following steps:

(1) mixing pediococcus pentosaceus and lactobacillus plantarum to obtain a composite strain;

(2) cleaning plant raw materials, adding water, adjusting the pH value to 3.5-4.0, sterilizing at 60 ℃ for 30min, cooling to room temperature, inoculating the composite strain, and fermenting to obtain the plant enzyme.

According to the method for preparing the plant enzyme, disclosed by the embodiment of the invention, the plant raw materials are subjected to mixed fermentation by adopting pediococcus pentosaceus and lactobacillus plantarum, the fruit aroma and the sweet aroma of a fermented product are obviously improved, the peculiar smell and the sour taste are obviously reduced, and the bioactive components are obviously improved.

In addition, the method for preparing plant ferment provided by the above embodiments of the present invention may further have the following additional technical features:

alternatively, in the step (1), the pediococcus pentosaceus and the lactobacillus plantarum are respectively activated and cultured in an expanding way to a logarithmic growth phase (cultured for 2-3 days at 28 ℃), and mixed after counting; mixing to obtain a mixture with a total bacteria concentration of 5 × 10 or more⁶The ratio of the number of the pediococcus pentosaceus to the CFU/mL bacterial liquid is 20-80%.

Optionally, in the step (2), the plant material is grape, grapefruit or edible seaweed.

Optionally, in the step (2), the fermentation temperature is 20-30 ℃, and the fermentation time is 3-15 days. During fermentation, the mixture is kept still without stirring.

Optionally, in the step (2), the initial number of the composite bacterial species is greater than or equal to 5 × 10⁴CFU/mL。

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Detailed Description

The technical solution of the present invention is illustrated by specific examples below. It is to be understood that one or more method steps mentioned in the present invention do not exclude the presence of other method steps before or after the combination step or that other method steps may be inserted between the explicitly mentioned steps; it should also be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Moreover, unless otherwise indicated, the numbering of the various method steps is merely a convenient tool for identifying the various method steps, and is not intended to limit the order in which the method steps are arranged or the scope of the invention in which the invention may be practiced, and changes or modifications in the relative relationship may be made without substantially changing the technical content.

In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the invention have been shown, it should be understood that the invention may be embodied in various forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The test materials adopted by the invention are all common commercial products and can be purchased in the market.

It should be noted that:

the invention carries out sensory inspection on the flavor according to relevant documents and the specification standard of ISO 8589. 5mL of the sample was collected, and 5 to 10 men and women (23 to 30 years old) who had experience in food sensory evaluation were invited to form an evaluation panel to evaluate the fishy smell, the mold smell, the grass smell, the sour smell, the floral smell, the sweet smell, and the fruity smell. To reduce the subjectivity of the evaluation, each process is encoded with a random number. The score range is 1-10 points, wherein 1 point is that the related odor is weak, and 10 points are that the related odor is strong. The evaluation results were expressed as average scores of sensory evaluators.

The invention adopts HPLC to measure the content of resveratrol which is an active ingredient. 5g of the fermented sample was added to 60mL of 4M hydrochloric acid solution and placed in a 90 ℃ water bath for 90 min. The residue was then filtered through a filter paper, rotary evaporated to dryness at 50 ℃ and 10mL of 50% methanol was added. The column was InertSustain C185 μm 4.6X 250mm as determined by High Performance Liquid Chromatography (HPLC). The mobile phase is acetonitrile: water 30: 70, the flow rate is 1.0mL/min, the detection wavelength is 306nm, and the column temperature is 40 ℃; the amount of sample was 10. mu.L.

The invention adopts spectrophotometry to determine the total phenol content: taking 0.1g of gallic acid, dissolving with 50mL of distilled water, and diluting to 100mL to obtain a gallic acid standard stock solution with a mass concentration of 1000 mg/mL. Weighing 0, 100, 200, 300, 400, 500 and 600 mu L of standard stock solution in a 10mL volumetric flask respectively, fixing the volume to the scale with distilled water, and preparing a series of standard solutions with mass concentrations of 0, 10, 20, 30, 40, 50 and 60 mg/mL. 1mL of the standard solution was put in a 10mL volumetric flask, and 1mL of the furin phenol reagent and 3mL of 7.5% Na were added respectively₂CO₃The solution is mixed evenly and reacted for 2h in a dark place. Measuring absorbance at 765nm wavelength, performing 3 parallel tests for each concentration, averaging, and drawing a standard curve to obtain a regression equation of absorbance value and gallic acid standard solution concentration. Taking 1mL of centrifuged grapefruit and seaweed fermentation supernatant, adding 1mL of folinol reagent and 3mL of 7.5% Na₂CO₃The solution is mixed evenly and reacted for 2h in a dark place. And (3) measuring the absorbance at the wavelength of 765nm, and substituting the absorbance into a regression equation to obtain the total phenol content.

The invention will now be described with reference to specific examples, which are intended to be illustrative only and not to be limiting in any way.

Example 1

Freeze-drying 100g fresh grape, grinding into powder, adding into 250mL conical flask, adding water to 150mL, adjusting initial pH to 4.0, sterilizing, inoculating Pediococcus pentosaceus and Lactobacillus plantarum (Pediococcus pentosaceus accounts for 50% of composite strain, total inoculum size is 1%, and initial strain concentration of composite strain is 5 × 10)⁴CFU/mL), fermenting at 28 ℃ for 10d to obtain a grape fermentation liquid. The flavour evaluation results showed a pronounced sweet (4.9 points) and fruity (3.6 points) notes after fermentation, as well as a moderate sour taste (3.3 points) with a resveratrol peak area of 75150umu (table 1). Compared with the fermentation only by using lactobacillus plantarum, the fermentation by using the composite strain of the embodiment has the advantages that the sweet flavor is increased by 32.4%, the fruit flavor is increased by 24.14%, the sour flavor is reduced by 17.5% compared with the fermentation only by using lactobacillus plantarum, the overall flavor is obviously improved, and the resveratrol content is increased by 192.98% (table 1).

TABLE 1 grape raw materials (comparative example 1) and Lactobacillus plantarum fermentations (comparative example 2) and Complex bacteria fermentations of this example grape odor and resveratrol content

Example 2

Grinding 1g of freeze-dried edible seaweed into powder, placing into a 250mL conical flask, adding water to 100mL, adjusting initial pH to 4.0, sterilizing, inoculating Pediococcus pentosaceus and Lactobacillus plantarum (Pediococcus pentosaceus accounts for 25% of the composite strain, total inoculum size is 5%, and initial strain concentration of composite strain is 2.5 × 10)⁵CFU/mL), fermenting for 3d at 28 ℃ to obtain seaweed fermentation liquor. The flavour assessment results showed that it had a strong sweet flavour (4.1 points), a moderate sour flavour (3.1), as well as a slight fishy flavour (2.0 points), a musty flavour (2.1 points), a grassy flavour (2.6 points) and a floral flavour (2.5), with a total polyphenol content flavour of 58.88 μ g/g. Compared with the seaweed fermented only by lactobacillus plantarum, the sweet taste and the flower fragrance are increased by 51.58% and 25%, the mould soil taste is reduced by 60.37%, the green grass taste is reduced by 30.43%, the fishy smell is reduced by 25.9%, the sour taste is reduced by 16.2%, and the overall flavor is obviously improved; the total polyphenol content increased by 17.76 (table 2).

TABLE 2 seaweed smell and Total phenol content of seaweed Material (comparative example 3) fermented by Lactobacillus plantarum (comparative example 4) and Complex bacteria of this example

Example 3

Freeze-drying fresh fructus Citri Grandis 100g, grinding into powder, adding into 250mL conical flask, adding water to 150mL, adjusting initial pH to 4.0, sterilizing, inoculating Pediococcus pentosaceus and Lactobacillus plantarum (Pediococcus pentosaceus accounts for 75% of composite strain, total inoculum size is 5%, and initial strain concentration of composite strain is 2.5 × 10%⁵CFU/mL), and fermenting for 3 days at 28 ℃ to obtain the grapefruit fruit fermentation liquor. Flavor evaluation result display toolHas strong sour taste (4.7 points) and fruity flavor (4.0 points), and moderate sweet flavor (3.3 points), and has total polyphenol content of 26.92 μ g/g. Compared with the grapefruit juice fermented only by lactobacillus plantarum, the sour taste of the grapefruit juice fermented by the compound strain is reduced by 21.8%, the sweet flavor is improved by 120%, the fruit flavor is improved by 60%, and the total polyphenol content is improved by 122.47% (table 3).

TABLE 3 measurement of smell and Total phenols content of grapefruit Material (comparative example 5) by Lactobacillus plantarum fermentation (comparative example 6) and Complex bacteria fermentation of the present example

Comparative example 1

Grapes that have not undergone any fermentation. 100g of fresh grapes are crushed and added into a 250mL conical flask, water is added to 150mL, the initial pH is 4.0, 1% distilled water (simulated inoculation volume) is added after sterilization, and the grapes are placed for 3d at 28 ℃. The flavor evaluation results showed that it had a slightly sour taste (0.7 point), a strong fruit note (5.9 points) and a sweet note, with a resveratrol peak area of 6717umu (table 1).

Comparative example 2

Grapes fermented only by lactobacillus plantarum. Taking 100g of fresh grapes, mashing the grapes, adding the grapes into a 250mL conical flask, adding water to 150mL, adjusting the initial pH to 4.0, inoculating 1% lactobacillus plantarum after sterilization, and fermenting for 10d at 28 ℃ to obtain grape fermentation liquid. The flavor evaluation result shows that the vinegar beverage has strong sour taste (4.0 points), while sweet flavor (3.7 points) and fruit flavor (2.9 points) are not obvious, and the whole flavor is not good; the peak area of resveratrol detected by HPLC was 25650umu (Table 1).

Comparative example 3

The seaweed is not fermented by any bacteria. Freeze-drying 1g of fresh Haematococcus, grinding into powder, adding into a 250mL conical flask, adding water to 100mL, adjusting initial pH to 4.0, sterilizing, and standing at 28 deg.C for 3d to obtain Sargassum liquid. The flavor evaluation result shows that the product has strong moldy soil flavor (7.5 points), obvious fishy smell (5.5 points), slightly obvious green grass flavor (4.7 points) and sweet flavor (4.5 points) and unobvious flower flavor (2.3 points); the total polyphenol content measured by the Folin phenol method showed a total amount of 33.64 μ g/g (Table 2).

Comparative example 4

Seaweed fermented only with lactobacillus plantarum. Freeze-drying 1g of fresh Haematococcus pluvialis, grinding into powder, adding into a 250mL conical flask, adding water to 100mL, adjusting initial pH to 4.0, sterilizing, inoculating 5% Lactobacillus plantarum, and fermenting at 28 deg.C for 3d to obtain seaweed fermentation liquid. The flavor evaluation result shows that the tea has obvious moldy soil flavor (5.3 points), moderate green grass flavor (3.3 points) and sour flavor (3.1 points), and weak fishy smell (2.7 points), flower fragrance (2.0 points) and sweet fragrance (2.7 points); the total polyphenol content was 50. mu.g/g (Table 2).

Comparative example 5

The shaddock is not added with any bacteria for fermentation. Freeze-drying 100g of fresh pomelo, grinding into powder, adding into a 250mL conical flask, adding water to 150mL, adjusting the initial pH to 4.0, sterilizing, and standing at 28 deg.C for 3d to obtain pomelo juice. The flavor evaluation results showed that the fruit aroma (score 6.6) was intense with the sweet aroma (score 4.7), with moderate sour taste (score 2.7), and the total polyphenol content was 13.46 μ g/g (table 3).

Comparative example 6

Fructus Citri Grandis fermented by Lactobacillus plantarum. Freeze-drying 100g of fresh pomelo, grinding into powder, adding into a 250mL conical flask, adding water to 150mL, adjusting the initial pH to 4.0, sterilizing, inoculating 5% lactobacillus plantarum, and fermenting at 28 deg.C for 3d to obtain pomelo fermentation liquor. The flavor evaluation result shows that the vinegar is strong in sourness (5.5 points), and has moderate fruit aroma (2.5 points) and sweet aroma (1.5 points) which are weak; the total polyphenol content was 12.1. mu.g/g (Table 3).

In conclusion, according to the embodiment of the invention, by adopting a strain mode of mixed fermentation of pediococcus pentosaceus and lactobacillus plantarum, the fruit juice aroma and the sweet aroma after fermentation are obviously increased, the fermentation smell, the musty taste, the sour taste, the fishy smell and the like are obviously reduced, meanwhile, the content of active ingredients in the fermentation product is obviously improved, the flavor and the content of the active ingredients are obviously superior to those of the single lactobacillus plantarum fermentation, and the lactobacillus pentosaceus is widely suitable for fermentation of various plants.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above should not be understood to necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples described in this specification can be combined and combined by those skilled in the art.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.