Method for improving activity of metabolic esterase of lactic acid bacteria

Abstract

The invention discloses a method for improving the activity of metabolic esterase of lactic acid bacteria. The method comprises the step of improving the activity of metabolic esterase of lactic acid bacteria by combining cold treatment of a lactic acid bacteria strain culture solution with a method of adding metal ions into a pre-cultured culture medium. The method specifically comprises the following steps: inoculating an activated strain into 100mL of an MRS culture medium according to the inoculation amount of 3% (v / v), then adding metal ions, uniformly vibrating, and culturing at 10 DEG C for 5 hours. By adopting the technical scheme, the activity of metabolic esterase of lactic acid bacteria can be remarkably improved, the fermentation efficiency is improved, and new flavor substances can be generated when the lactobacillus is applied to fermented dairy products, so that new product types are developed.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and more particularly to a method for improving the activity of lactic acid bacteria metabolic esterase. Background Art

[0002] Esterase is a class of enzymes that catalyze ester hydrolysis and ester synthesis reactions, and can synthesize esterification products with different flavors in different fermented foods.

[0003] The characteristics of aroma production by fermentation technology are unique aroma type, but low aroma intensity and low aroma substance yield, while the characteristics of aroma production by enzyme engineering technology are high flavor intensity, but single aroma type and easy to produce bad flavor. If these two technologies are combined, the aroma production rate can be accelerated and the aroma intensity can be improved under the premise of ensuring the characteristic flavor. When the esterase content of the lactic acid bacteria fermentation system increases, milk fat is enzymatically hydrolyzed to produce various saturated fatty acids, unsaturated fatty acids, keto acids and hydroxy acids. Most fatty acids are C4-C14 even-chain fatty acids, with a high degree of aroma contribution, showing a unique enzymatic flavor. They are the main aroma substances of milk fat enzymatic flavor, and enzymatic hydrolysis can significantly improve the flavor of dairy products. At the same time, a large number of free fatty acids can participate in the complex biochemical reaction process of lactic acid bacteria fermentation, produce some new flavor substances, such as methyl ketones and flavor esters, etc., forming a more complex and rich natural flavor system. Keto acids such as β-keto acids are decarboxylated in organisms to produce various methyl ketones, such as 2-heptanone, 2-nonanone, 2-undecanone and 2-tridecanone. Hydroxylic acids undergo dehydration and other reactions in the body to cyclize into various δ-lactones and γ-lactones. These methyl ketones and lactones are the key flavor substances in the formation of frankincense.

[0004] By adjusting the culture conditions and the composition of the culture medium, the content of lactic acid bacteria metabolic esterase can be increased and applied to fermented dairy products to produce new flavor substances, thereby developing new product types. Therefore, how to provide a simple method for increasing the activity of lactic acid bacteria metabolic esterase is a technical problem that those skilled in the art need to solve urgently. Summary of the invention

[0005] In view of this, the present invention provides a method for improving the activity of lactic acid bacteria metabolic esterase, which adopts cold stress treatment combined with the addition of divalent metal ions to increase the activity of lactic acid bacteria metabolic esterase, aiming to develop fermented dairy products with new flavors.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for improving the activity of lactic acid bacteria metabolic esterase, which improves the activity of lactic acid bacteria metabolic esterase by cold treatment of lactic acid bacteria strain culture solution and adding metal ions into the culture medium.

[0008] Preferably, the cold treatment is to pre-culture the activated lactic acid bacteria strain culture solution at 10°C for 5 hours and then ferment it at 42°C.

[0009] The beneficial effects brought about by the above optimization are: the present invention uses cold stress to activate lactic acid bacteria strains, which can improve the metabolic activity of lactic acid bacteria and shorten the activation time.

[0010] Preferably, the activation is to inoculate the frozen lactic acid bacteria into 100 mL of MRS medium at a rate of 5% (v / v), shake well, culture at 37°C for 24 h, subculture and activate 3 times to ensure that the number of viable bacteria is 10 8 CFU / mL, for future use.

[0011] The beneficial effects of the above optimization are: the above nutrient medium, inoculation concentration, activation temperature and time are selected to achieve the optimal growth conditions of lactic acid bacteria, complete pre-culture, and ensure the number of viable bacteria. 。

[0012] Preferably, the pre-culture is to inoculate the activated strain into 100 mL of MRS medium at an inoculum volume of 3% (v / v), shake well, and culture at a constant temperature of 37°C.

[0013] The beneficial effects brought about by the above optimization are: suitable inoculation amount and fermentation temperature can improve fermentation efficiency.

[0014] Preferably, the MRS medium comprises the following components:

[0015] 5g beef extract, 5g peptone, 10g tryptone, 5g yeast powder, 1mL Tween 80, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.8g magnesium sulfate, and 0.25g manganese sulfate.

[0016] Preferably, the MRS medium is prepared by the following method:

[0017] The components of the MRS medium were mixed, and then the volume was made up to 1000 mL with distilled water, the pH was adjusted to 6.0, and sterilized at 121° C. for 15 min.

[0018] The beneficial effects of the above optimization are: adjusting the culture conditions and culture medium composition, improving the activity of lactic acid bacteria metabolic esterase, and increasing the content of lactic acid bacteria metabolic esterase. 。

[0019] Preferably, the metal ion is a divalent metal ion.

[0020] Preferably, the divalent metal ions are any one or more mixtures of magnesium ions, calcium ions, iron ions and zinc ions.

[0021] The beneficial effects of the above optimization are: magnesium ions, calcium ions, divalent iron ions, and zinc ions have a significant promoting effect on the improvement of enzyme activity. 。

[0022] Preferably, the added amount of each ion in the divalent metal ions is: 6 mmol / L of magnesium ions, 2.4 mmol / L of calcium ions, 2 mmol / L of divalent iron ions, and 1.6 mmol / L of zinc ions.

[0023] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention adopts cold stress treatment combined with the addition of divalent metal ions, which can significantly improve the metabolic esterase activity of lactic acid bacteria and enhance the fermentation efficiency. It is applied to fermented dairy products and can produce new flavor substances, thereby developing new product types. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 It is a technical flow chart of the present invention;

[0027] Figure 2 This is a graph showing the effect of adding different concentrations of iron ions on the activity of lactic acid bacteria metabolic esterase;

[0028] Figure 3 This is a graph showing the effect of adding different concentrations of zinc ions on the activity of lactic acid bacteria metabolic esterase;

[0029] Figure 4 This is a graph showing the effect of adding different concentrations of magnesium ions on the activity of lactic acid bacteria metabolic esterase;

[0030] Figure 5 This graph shows the effect of adding different concentrations of calcium ions on the activity of lactic acid bacteria metabolic esterase. DETAILED DESCRIPTION

[0031] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] Example 1

[0033] This embodiment provides a method for improving the activity of lactic acid bacteria metabolic esterase, comprising:

[0034] (1) Inoculate the frozen lactic acid bacteria into 100 mL of MRS medium at a 5% (v / v) inoculum, shake well, and culture at 37°C for 24 h. Subculture and activate three times to ensure that the number of viable bacteria is 10 8 CFU / mL, reserve;

[0035] (2) The activated strain was inoculated into 100 mL of MRS medium at an inoculum size of 3% (v / v), and then divalent metal ions were added, shaken, cultured at 10° C. for 5 h, and then fermented at 42° C.

[0036] The MRS medium was prepared by the following method: 5 g beef extract, 5 g peptone, 10 g tryptone, 5 g yeast powder, 1 mL Tween 80, 20 g glucose, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g sodium acetate, 0.8 g magnesium sulfate, and 0.25 g manganese sulfate were mixed, then the volume was adjusted to 1000 mL with distilled water, the pH was adjusted to 6.0, and sterilized at 121°C for 15 min.

[0037] The optimal parameters in the method of the present invention are determined by experiments. The specific experiments are as follows:

[0038] Test 1

[0039] (1) Preparation of crude esterase solution of lactic acid bacteria fermentation system. The strain used in the study was Lactobacillus paracasei (hereinafter the same). 10 g of bacterial fermentation solution was placed in a centrifuge tube and centrifuged at 4°C and 10,000 g for 3 min. The precipitated bacteria were washed with pH 7.5 phosphate buffer and then ultrasonically disrupted the cell wall. The cycle lasted for 10 min, with 10 s of disruption and 15 s of rest. The disruption solution was centrifuged at 4°C and 10,000 g for 15 min. The supernatant obtained was the crude enzyme solution.

[0040] (2) Determination of esterase activity: the amount of enzyme required to hydrolyze 4 mL of 3% tributyrin emulsion at 40°C for 15 min is 1 unit of enzyme activity;

[0041] (3) Adding divalent metal ions to the pre-culture medium can improve the activity of esterase, among which magnesium ions, calcium ions, divalent iron ions, and zinc ions have a significant promoting effect on the improvement of enzyme activity. The specific experimental results are shown in the table below:

[0042] Effect of metal ion addition on enzyme activity (n=3, M±SD)

[0043]

[0044] Note: "+" represents an increase in enzyme activity compared with the blank control group, and "-" represents a decrease in enzyme activity compared with the blank control group

[0045] (4) The effect of adding different concentrations of magnesium ions on the activity of lactic acid bacteria esterase is shown in Figure 4 When the magnesium ion concentration was 6 mmol / L, the lactic acid bacteria esterase activity was 4.318 U / mL under this condition, which was 14.2% higher than that before optimization (P<0.05).

[0046] (5) The activated strain culture fluid was pre-cultured at 10°C for 5 h and then fermented at 42°C. Cold stress significantly increased the metabolic activity of lactic acid bacteria, which was 22.7% higher than that of the control sample.

[0047] (6) Cold stress treatment combined with the addition of divalent metal ions can significantly increase the activity of esterases in lactic acid bacteria. After cold stress at 10°C for 5 h, the addition of 2.4 mmol / L calcium ions, 2 mmol / L divalent iron ions and 1.6 mmol / L zinc ions increased the esterase activity by 52.6% (P<0.05).

[0048] Test 2

[0049] (1) Preparation of crude esterase solution of lactic acid bacteria fermentation system: 10 g of bacterial fermentation solution was placed in a centrifuge tube, centrifuged at 4°C and 10,000 g for 3 min, the precipitated bacteria were washed with pH 7.5 phosphate buffer and then ultrasonically disrupted the cell wall, with a cycle of 10 s of disruption and 15 s of rest, which lasted for 10 min in total, and the disrupted solution was centrifuged at 4°C and 10,000 g for 15 min to obtain the supernatant as the crude enzyme solution;

[0050] (2) Determination of esterase activity: the amount of enzyme required to hydrolyze 4 mL of 3% tributyrin emulsion at 40°C for 15 min is 1 unit of enzyme activity;

[0051] (3) Adding divalent metal ions to the culture medium can improve the activity of esterase, among which magnesium ions, calcium ions, divalent iron ions and zinc ions have a significant promoting effect on the improvement of enzyme activity. The specific experimental results are shown in the table below:

[0052] Effect of metal ion addition on enzyme activity (n=3, M±SD)

[0053]

[0054] Note: "+" represents an increase in enzyme activity compared with the blank control group, and "-" represents a decrease in enzyme activity compared with the blank control group

[0055] (4) The effects of different concentrations of calcium ions on the activity of lactic acid bacteria esterase are shown in Figure 5 When the amount of calcium ion added was 2.4mmol / L, the activity of lactic acid bacteria esterase was 4.437U / mL under this condition, which was 17.3% higher than that before optimization (P<0.05);

[0056] (5) The activated strain culture solution was pre-cultured at 10°C for 5 h and then fermented at 42°C. Cold stress significantly increased the metabolic activity of lactic acid bacteria, which was 22.7% higher than that of the control sample.

[0057] (6) Cold stress treatment combined with the addition of divalent metal ions can significantly increase the activity of esterases in lactic acid bacteria. After cold stress at 10°C for 5 h, the addition of 2.4 mmol / L calcium ions, 2 mmol / L divalent iron ions and 1.6 mmol / L zinc ions increased the esterase activity by 52.6% (P<0.05).

[0058] Test 3

[0059] (1) Preparation of crude esterase solution of lactic acid bacteria fermentation system: 10 g of bacterial fermentation solution was placed in a centrifuge tube, centrifuged at 4°C and 10,000 g for 3 min, the precipitated bacteria were washed with pH 7.5 phosphate buffer and then ultrasonically disrupted the cell wall, with a cycle of 10 s of disruption and 15 s of rest, which lasted for 10 min in total, and the disrupted solution was centrifuged at 4°C and 10,000 g for 15 min to obtain the supernatant as the crude enzyme solution;

[0060] (2) Determination of esterase activity: the amount of enzyme required to hydrolyze 4 mL of 3% tributyrin emulsion at 40°C for 15 min is defined as 1 unit of enzyme activity.

[0061] (3) Adding divalent metal ions to the culture medium can improve the activity of esterase, among which magnesium ions, calcium ions, divalent iron ions and zinc ions have a significant promoting effect on the improvement of enzyme activity. The specific experimental results are shown in the table below:

[0062] Effect of metal ion addition on enzyme activity (n=3, M±SD)

[0063]

[0064] Note: "+" represents an increase in enzyme activity compared with the blank control group, and "-" represents a decrease in enzyme activity compared with the blank control group

[0065] (4) On this basis, an orthogonal experiment was carried out, and the final optimized culture medium formula was as follows: the addition amount of magnesium ions was 6mmol / L, the addition amount of calcium ions was 2.4mmol / L, the addition amount of divalent iron ions was 2mmol / L, and the addition amount of zinc ions was 1.6mmol / L; under this condition, the activity of lactic acid bacteria esterase was 4.998U / mL, which was 34.5% higher than that before optimization (P<0.05).

[0066] (5) The activated strain culture fluid was pre-cultured under different temperature conditions and then fermented at 42°C. Cold stress significantly increased the metabolic activity of lactic acid bacteria, which was 22.7% higher than that of the control sample.

[0067] Effect of cold stress culture of lactic acid bacteria on esterase activity (n=3, M±SD)

[0068]

[0069]

[0070] (6) Cold stress treatment combined with the addition of divalent metal ions can significantly increase the activity of esterases in lactic acid bacteria. After cold stress at 10°C for 5 h, the addition of 2.4 mmol / L calcium ions, 2 mmol / L divalent iron ions and 1.6 mmol / L zinc ions increased the esterase activity by 52.6% (P<0.05).

[0071] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0072] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for improving the activity of lactic acid bacteria metabolic esterase, characterized in that: include: The activity of metabolic esterase of lactic acid bacteria is improved by cold treatment of lactic acid bacteria culture solution and adding metal ions into pre-culture medium.

2. A method for improving the activity of lactic acid bacteria metabolic esterase according to claim 1, characterized in that: The cold treatment is to pre-culture the activated lactic acid bacteria strain at 10° C. for 5 hours, and then ferment at 42° C. for 10 hours.

3. A method for improving the activity of lactic acid bacteria metabolic esterase according to claim 2, characterized in that: The activation step is to inoculate the frozen lactic acid bacteria into 100 mL of MRS medium at a 5% (v / v) inoculation volume, shake well, culture at 37°C for 24 hours, subculture and activate for 3 times to ensure that the number of viable bacteria is 10 8 CFU / mL, for future use.

4. A method for improving the activity of lactic acid bacteria metabolic esterase according to claim 3, characterized in that: The pre-culture is specifically to inoculate the activated strain into 100 mL of MRS medium at an inoculum volume of 3% (v / v), then add metal ions, shake well, and culture at 10° C. for 5 hours.

5. A method for improving the activity of lactic acid bacteria metabolic esterase according to claim 4, characterized in that: The MRS medium includes the following components: 5g beef extract, 5g peptone, 10g tryptone, 5g yeast powder, 1mL Tween 80, 20g glucose, 2g dipotassium hydrogen phosphate, 2g diammonium hydrogen citrate, 5g sodium acetate, 0.8g magnesium sulfate, and 0.25g manganese sulfate.

6. A method for improving the activity of lactic acid bacteria metabolic esterase according to claim 5, characterized in that: The MRS culture medium is prepared by the following method: The components of the MRS medium were mixed, and then the volume was adjusted to 1000 mL with water, the pH was adjusted to 6.0, and sterilized at 121° C. for 15 min.

7. The method for improving the activity of lactic acid bacteria metabolic esterase according to claim 1, characterized in that: The metal ions are divalent metal ions.

8. A method for improving the activity of lactic acid bacteria metabolic esterase according to claim 7, characterized in that: The divalent metal ions are any one or more of magnesium ions, calcium ions, iron ions and zinc ions.

9. A method for improving the activity of lactic acid bacteria metabolic esterase according to claim 8, characterized in that: The added amount of each ion in the divalent metal ions is: 6 mmol / L magnesium ion, 2.4 mmol / L calcium ion, 2 mmol / L divalent iron ion, and 1.6 mmol / L zinc ion.

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