A method for increasing the glycosidase activity of intact cells of brewing lactic acid bacteria during fermentation

Abstract

A method for increasing the glycosidase activity of intact cells of brewing lactic acid bacteria in fermentation, the method comprising strain activation, strain expansion culture, strain induction culture, strain adaptation culture and fermentation culture steps, characterized in that different carbon sources are added in the strain activation, strain expansion culture and strain induction culture steps, ultrasonic treatment is used in the strain induction culture and fermentation culture, and finally fermentation parameters are regulated in the fermentation culture, so as to increase the glycosidase activity of intact cells of brewing lactic acid bacteria in fermentation. Through this method, the different glycosidase activities of intact cells of brewing lactic acid bacteria in fermentation are greatly improved, the degradation of glycoside substances in fermentation is promoted, the aroma of fruit wine is greatly increased, and the overall quality of fruit wine is improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for increasing the glycosidase activity of intact cells of brewing lactic acid bacteria during fermentation. Background Art

[0002] High-quality fruit wine is not only reflected in appearance and taste, but also in aroma. Aroma is one of the key factors affecting the quality of fruit wine. The aroma that contributes greatly to the flavor of fruit wine can be divided into three categories according to its source: variety aroma, brewing aroma and aging aroma. There are two kinds of aroma substances in wine-making fruits: free and bound. Free aroma substances, i.e. volatile aroma substances, can be directly felt by people, while a large number of bound aroma substances in the form of glycosides cannot be felt by people. Only after degradation and release of free aroma substances can people feel them. Glycosides mainly exist in the form of monosaccharides, disaccharides and rare trisaccharides, mainly β-D-pyranoglucoside, 6-O-α-L-arabinofuranosyl-β-D-pyranoglucoside, 6-O-α-L-rhamnopyranosyl-β-D-pyranoglucoside, 6-O-α-L-pyranose rhamnosyl-β-D-pyranoglucoside, 6-O-α-L-furanose celery glycoside-β-D-pyranoglucoside, etc.

[0003] Glycosidase hydrolysis is the main way to degrade glycosides to produce aroma substances in fruit wine brewing, and Oenococcus oeni, which dominates the secondary fermentation of fruit wine, is the main microorganism that produces glycosidases during fermentation. However, the glycosidases produced by Oenococcus oeni are basically intracellular enzymes. The intact cells have a certain enzyme activity, but the activity is low, so that most of the bound aroma substances-glycosides in the raw materials cannot be degraded, making the fruit wine aroma bland and the quality average. Therefore, improving the glycosidase activity of intact cells of Oenococcus oeni during fermentation and increasing the degradation of bound aroma substances-glycosides during fermentation are important for increasing the aroma of fruit wine and improving the overall quality of fruit wine. Summary of the invention

[0004] Purpose of the invention: The technical problem to be solved by the present invention is to provide a method for increasing the glycosidase activity of whole cells of lactic acid bacteria in fermentation in view of the shortcomings of the prior art, that is, the defects and deficiencies of the glycosidase activity of whole cells of lactic acid bacteria in fruit wine fermentation. The method can greatly increase the glycosidase activity of whole cells of lactic acid bacteria in fermentation, and produce high-quality fruit wine with strong aroma.

[0005] In order to solve the above technical problems, the present invention discloses a method for increasing the glycosidase activity of intact cells of brewing lactic acid bacteria in fermentation, the method comprising the steps of strain activation, strain expansion culture, strain induction culture, strain adaptation culture and fermentation culture. By adding different carbon sources in the steps of strain activation, strain expansion culture and strain induction culture, using ultrasonic treatment in strain induction culture and fermentation culture, and finally regulating fermentation parameters in fermentation culture, the glycosidase activity of intact cells of brewing lactic acid bacteria in fermentation is increased.

[0006] in,

[0007] (1) Activation of strains: streaking the frozen lactic acid bacteria on a solid culture medium and culturing at 20-25° C. for 10-15 days, wherein the carbon source of the solid culture medium is 5-8 g / L glucose and 100-150 mL / L Jerusalem artichoke pulp;

[0008] (2) Expanding the culture of the strain: picking a single colony after streaking culture in step (1), and expanding the culture in a liquid culture medium at 20-25° C. for 7-10 days; the carbon source of the liquid culture medium is: 8-10 g / L cellobiose, 100-150 mL / L Jerusalem artichoke pulp;

[0009] (3) Induction culture of bacterial strains: the culture fluid obtained after the expanded culture in step (2) is centrifuged at 5000g for 5 minutes to obtain bacterial bodies, and the bacterial bodies are inoculated into induction liquid culture medium A for primary induction culture; the bacterial bodies obtained by centrifuging the primary induction culture fluid again at 5000g for 5 minutes are inoculated into induction liquid culture medium B for secondary induction culture; ultrasonic treatment is performed at 50-100w for 15-20min on the second day of the primary induction culture and the second induction culture respectively; the carbon source of the induction liquid culture medium A is Jerusalem artichoke extract and a mixture of monosaccharides; the carbon source of the induction liquid culture medium B is Jerusalem artichoke extract and a mixture of disaccharides;

[0010] (4) strain adaptation culture: the bacteria obtained by centrifuging the secondary induction culture solution in step (3) at 5000 g for 5 min were inoculated into the adaptation medium and cultured at 18-20° C. for 4-5 days;

[0011] (5) Fermentation culture: The adapted culture solution obtained after the adaptation culture in step (4) is centrifuged at 5000g for 5 minutes, and the bacterial cells are inoculated into fruit wine with a total sugar content of 10-20 g / L and a SO2 content of less than 30 mg / L at an inoculum amount of 8-10 g / L of wet bacterial cells at 18-20°C for 15-20 days. The fermentation is performed by ultrasonic treatment at 100-150w for 20-30 minutes on the 5th and 10th days of the fermentation culture.

[0012] Wherein, in step (1), the brewing lactic acid bacteria include any one of Oenococcus oeni and Leucanostoc.

[0013] Preferably, as described in Example 1, when making wine, in step (1), the winemaking lactic acid bacteria is Oenococcus oeni VP41 (purchased from Shanghai Jietu Biotechnology Co., Ltd.).

[0014] Preferably, as described in Example 2, when making blueberry wine, in step (1), the winemaking lactic acid bacteria is Oenococcus oeni 31MBR (purchased from Shanghai Jietu Biotechnology Co., Ltd.).

[0015] Wherein, in step (1), the activation conditions of the bacterial strain are preferably streaking culture at 20°C for 15 days in Example 1, and preferably streaking culture at 25°C for 10 days in Example 2.

[0016] Wherein, in step (1), the solid culture medium has a carbon source of 5-8 g / L glucose, 100-150 mL / L Jerusalem artichoke pulp, and other ingredients of 5 g / L yeast extract, 8 g / L peptone, 0.15 g / L MgSO4·7H2O, 0.02 g / L MnSO4·4H2O, pH 4.3-4.5, and 20 g / L agar.

[0017] Preferably, as described in Example 1, when making wine, in step (1), the solid culture medium is 5 g / L glucose, 150 mL / L Jerusalem artichoke pulp, 5 g / L yeast extract powder, 8 g / L peptone, 0.15 g / L MgSO4·7H2O, 0.02 g / L MnSO4·4H2O, pH 4.3, and 20 g / L agar.

[0018] Preferably, as described in Example 2, when making blueberry wine, in step (1), the solid culture medium is 8 g / L glucose, 100 mL / L Jerusalem artichoke pulp, 5 g / L yeast extract powder, 8 g / L peptone, 0.15 g / L MgSO4·7H2O, 0.02 g / L MnSO4·4H2O, pH 4.3, and 20 g / L agar.

[0019] Specifically, the solid culture medium uses glucose as a carbon source in order to rapidly recover and grow the bacteria, and Jerusalem artichoke pulp is used as a carbon source in order to rapidly grow the bacteria and induce the expression of its glycosidase gene.

[0020] Wherein, in step (2), the expansion culture conditions of the strain are preferably expanded culture at 20°C for 10 days in Example 1, and preferably expanded culture at 25°C for 7 days in Example 2.

[0021] Wherein, in step (2), the liquid culture medium comprises 8-10 g / L of cellobiose, 100-150 mL / L of Jerusalem artichoke pulp, 5 g / L of yeast extract powder, 8 g / L of peptone, 0.15 g / L of MgSO4·7H2O, 0.02 g / L of MnSO4·4H2O, and a pH of 4.3-4.5.

[0022] Preferably, as described in Example 1, when making wine, in step (2), the liquid culture medium comprises 8 g / L of cellobiose, 150 mL / L of Jerusalem artichoke pulp, 5 g / L of yeast extract powder, 8 g / L of peptone, 0.15 g / L of MgSO4·7H2O, 0.02 g / L of MnSO4·4H2O, and pH 4.3.

[0023] Preferably, as described in Example 2, when making blueberry wine, in step (2), the liquid culture medium comprises 10 g / L of cellobiose, 100 mL / L of Jerusalem artichoke pulp, 5 g / L of yeast extract powder, 8 g / L of peptone, 0.15 g / L of MgSO4·7H2O, 0.02 g / L of MnSO4·4H2O, and pH 4.5.

[0024] Specifically, cellobiose and Jerusalem artichoke pulp are used as carbon sources in the liquid culture medium, with the purpose of inducing the expression of bacterial glycosidase genes and glycosidase proteins.

[0025] Among them, in step (1) and step (2), the Jerusalem artichoke pulp is prepared by mixing the washed Jerusalem artichoke and pure water in a weight ratio of 8:2 and beating the mixture to obtain the pulp.

[0026] Wherein, in step (3), the culture conditions of the first induction culture are: 18-20°C, culture for 2-3 days, and an ultrasonic treatment is performed on the second day, the treatment conditions are 50-100w, and the ultrasonic time is 15-20min; the culture conditions of the second induction culture are 18-20°C, culture for 3-4 days, and an ultrasonic treatment is performed on the second day, the treatment conditions are 50-100w, and the ultrasonic time is 15-20min.

[0027] Preferably, as described in Example 1, when making wine, in step (3), the primary induction culture is cultured at 18° C. for 3 days, followed by an ultrasonic treatment at 50 W for 20 min on the second day; the secondary induction culture is cultured at 18° C. for 4 days, followed by an ultrasonic treatment at 50 W for 20 min on the second day.

[0028] Preferably, as described in Example 2, when making blueberry wine, in step (3), the primary induction culture is cultured at 20°C for 2 days, followed by an ultrasonic treatment at 100W for 15 minutes on the second day; the secondary induction is cultured at 20°C for 3 days, followed by an ultrasonic treatment at 100W for 15 minutes on the second day.

[0029] Specifically, the ultrasonic treatment mainly utilizes ultrasound to stimulate cells, improve cell membrane permeability, promote the transport of substrates and degradation products, and thus improve the enzyme activity of intact bacterial cells.

[0030] Wherein, in step (3), the carbon source of the induction liquid culture medium A is a mixture of Jerusalem artichoke extract and monosaccharide, the weight ratio of the two is 1:5-1:6, the addition amount is 11-12 g / L, and the other components are yeast extract powder 5 g / L, peptone 8 g / L, MgSO4·7H2O 0.15 g / L, MnSO4·4H2O 0.02 g / L, pH 4.3-4.5; the carbon source of the induction liquid culture medium B is a mixture of Jerusalem artichoke extract and disaccharide, the weight ratio of the two is 1:7-1:8, the addition amount is 12-14 g / L, and the other components are yeast extract powder 5 g / L, peptone 8 g / L, MgSO4·7H2O 0.15 g / L, MnSO4·4H2O 0.02 g / L, pH 4.3-4.5.

[0031] Preferably, as described in Example 1, when making wine, in step (3), the carbon source of the induction liquid culture medium A is Jerusalem artichoke extract and a mixture of monosaccharides, the weight ratio of the two is 1:5, the addition amount is 11 g / L, and the other components are yeast extract powder 5 g / L, peptone 8 g / L, MgSO4·7H2O 0.15 g / L, MnSO4·4H2O 0.02 g / L, pH 4.3; the carbon source of the induction liquid culture medium B is Jerusalem artichoke extract and a mixture of disaccharides, the weight ratio of the two is 1:7, the addition amount is 12 g / L, and the other components are yeast extract powder 5 g / L, peptone 8 g / L, MgSO4·7H2O 0.15 g / L, MnSO4·4H2O 0.02 g / L, pH 4.3.

[0032] Preferably, as described in Example 2, when making blueberry wine, in step (3), the carbon source of the induction liquid culture medium A is Jerusalem artichoke extract and a mixture of monosaccharides, the weight ratio of the two is 1:6, the addition amount is 12 g / L, and the other components are yeast extract powder 5 g / L, peptone 8 g / L, MgSO4·7H2O 0.15 g / L, MnSO4·4H2O 0.02 g / L, pH 4.5; the carbon source of the induction liquid culture medium B is Jerusalem artichoke extract and a mixture of disaccharides, the weight ratio of the two is 1:8, the addition amount is 14 g / L, and the other components are yeast extract powder 5 g / L, peptone 8 g / L, MgSO4·7H2O 0.15 g / L, MnSO4·4H2O 0.02 g / L, pH 4.5.

[0033] Specifically, the Jerusalem artichoke extract extraction method is as follows: fresh Jerusalem artichoke is washed, sliced, soaked, dried, and crushed to obtain Jerusalem artichoke powder, the Jerusalem artichoke powder is mixed with water at a weight ratio of 1:10, ultrasonically extracted at 70-80°C for 30-45 minutes, and then rotary evaporated at 35°C and allowed to stand at 0-4°C to obtain crystals, and dried to obtain Jerusalem artichoke extract. The Jerusalem artichoke extract contains oligofructose of varying lengths.

[0034] Specifically, the carbon source in the induction liquid culture medium A is a mixture of Jerusalem artichoke extract and monosaccharide glycosides, the purpose of which is to induce and promote the expression and secretion of bacterial glucosidase and improve its activity.

[0035] Specifically, the carbon source in the induction liquid culture medium B uses a mixture of Jerusalem artichoke extract and disaccharide glycosides, the purpose of which is to induce and promote the expression and secretion of glycosidases required for the degradation of disaccharides by bacteria on the basis of improving the activity of glucosidase in intact cells of the bacteria, and to improve its activity.

[0036] Wherein, in step (4), the adapted culture conditions of the bacteria are preferably 18° C. for 5 days in Example 1, and 20° C. for 4 days in Example 2.

[0037] Wherein, in step (4), the adaptation medium comprises the following components: glucose 5-8 g / L, ethanol content 5-8% v / v, SO2 content 20-25 mg / L, grape skin glycoside extract 15-20 mL / L, tartaric acid 3-4 g / L, malic acid 1-2 g / L, K2HPO4 1-1.5 g / L, MgSO4·7H2O 0.1-0.15 g / L, MnSO4·H2O 0.02-0.04 g / L, yeast extract 1-1.8 g / L, and pH 3.5-3.8.

[0038] Preferably, as described in Example 1, when making wine, in step (4), the adaptation medium comprises the following components: 5 g / L glucose, 5% v / v ethanol content, 20 mg / L SO2 content, 15 mL / L grape skin glycoside extract, 3 g / L tartaric acid, 1 g / L malic acid, 1 g / L K2HPO4, 0.1 g / L MgSO4·7H2O, 0.02 g / L MnSO4·H2O, 1 g / L yeast extract, and pH 3.5.

[0039] Preferably, as described in Example 2, when making blueberry wine, in step (4), the adaptation medium comprises the following components: 8 g / L glucose, 8% v / v ethanol content, 25 mg / L SO2 content, 20 mL / L grape skin glycoside extract, 4 g / L tartaric acid, 2 g / L malic acid, 1.5 g / L K2HPO4, 0.15 g / L MgSO4·7H2O, 0.04 g / L MnSO4·H2O, 1.8 g / L yeast extract, and pH 3.8.

[0040] Specifically, the glucosidoside extract in the adaptation medium composition contains a large amount of monosaccharide and disaccharide.

[0041] Specifically, the main purpose of step (4) is to allow the bacteria to gradually adapt to the fruit wine fermentation environment in the simulated wine culture medium and maintain a high glycosidase activity.

[0042] Wherein, in step (5), the fermentation culture is inoculated with an inoculum amount of 8-10 g / L of wet bacteria into a fruit wine with a total sugar content of 10-20 g / L and a SO2 content of less than 30 mg / L, and fermented at 18-20°C for 15-20 days.

[0043] Preferably, as described in Example 1, when making wine, in step (5), the fermentation culture is inoculated with 8 g / L of wet bacteria until the total sugar content is 12 g / L and the SO2 content is 22 mg / L in fruit wine and fermented at 18°C ​​for 20 days.

[0044] Preferably, as described in Example 2, when making blueberry wine, in step (5), the fermentation culture is inoculated with 10 g / L of wet bacteria until the total sugar content is 19 g / L and the SO2 content is 29 mg / L, and fermented at 20°C for 15 days in fruit wine.

[0045] Wherein, in step (5), the fermentation culture is subjected to ultrasonic treatment for 20-30 minutes at 100-150W on the 5th day and the 10th day respectively.

[0046] Preferably, as described in Example 1, when making wine, in step (5), the fermentation culture is ultrasonically treated for 30 minutes at 100W on the 5th and 10th days respectively.

[0047] Preferably, as described in Example 2, when making blueberry wine, in step (5), the fermentation culture is ultrasonically treated for 20 minutes at 150W on the 5th and 10th days respectively.

[0048] Specifically, the ultrasonic treatment is mainly aimed at improving the permeability of cell membranes, promoting the transport of substrates and degradation products, and thus improving the enzyme activity of intact bacterial cells.

[0049] Wherein, in step (5), the fruit wine mainly includes grape wine, blueberry wine, apple wine and crabapple wine.

[0050] Preferably, as described in Example 1, in step (5), the fruit wine is grape wine.

[0051] Preferably, as described in Example 2, in step (5), the fruit wine is blueberry wine.

[0052] The monosaccharide glycoside is any one of arbutin and raspberry glycoside, and the disaccharide glycoside is a combination of any two of genipin gentianobioside, stevia bioside, and ophiopogon disaccharide, and the mass ratio thereof is 1:1.

[0053] Preferably, as described in Example 1, the monosaccharide glycoside is arbutin, and the disaccharide glycoside is genipin gentiobioside and stevioside, and the mass ratio of the two is 1:1.

[0054] Preferably, as described in Example 2, the monosaccharide glycoside is raspberry glycoside; the disaccharide glycoside is gentiana disaccharide and genipin gentian disaccharide, and the mass ratio of the two is 1:1.

[0055] Wherein, the glycosidase includes glucosidase, arabinosidase, rhamnosidase and apiosidase.

[0056] Beneficial effects:

[0057] The present invention greatly improves the activities of different glycosidases in intact cells of brewing lactic acid bacteria by optimizing the steps of strain activation, strain expansion culture, strain induction culture, strain adaptation culture and fermentation culture, promotes the degradation of glycoside substances during fermentation, greatly increases the aroma of fruit wine, and improves the overall quality of fruit wine. First, different carbon sources are added to culture in the steps of strain activation and strain expansion culture to enable the bacteria to recover and grow quickly, and induce the expression of bacterial glycosidase genes and glycosidase proteins; then, different carbon sources are used to induce culture of the bacteria in stages, and ultrasonic treatment is performed to greatly improve the expression, secretion and activity of different glycosidases in intact cells of the bacteria; finally, by strain adaptation culture and regulating fermentation parameters in fermentation culture, the best growth conditions are provided for the bacteria to maintain high activities of different glycosidases. DETAILED DESCRIPTION

[0058] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0059] The method for preparing Jerusalem artichoke pulp in the following embodiment is as follows: cleaned Jerusalem artichokes and purified water are mixed and evenly beaten in a weight ratio of 8:2 to prepare the pulp.

[0060] The Jerusalem artichoke extract extraction method in the following embodiment is as follows: fresh Jerusalem artichoke is washed, sliced, soaked, dried, and crushed to obtain Jerusalem artichoke powder, the Jerusalem artichoke powder is mixed with water at a weight ratio of 1:10, ultrasonically extracted at 70°C to 80°C for 30min-45min, and then rotary evaporated at 35°C and allowed to stand at 0°C to 4°C to obtain crystals, and dried to obtain Jerusalem artichoke extract. The Jerusalem artichoke extract contains oligofructose of varying lengths.

[0061] The monosaccharide and disaccharide glycosides described in the following examples were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0062] The method for determining bacterial glycosidase activity in the following examples is as follows: refer to Li Yahui et al., Study on β-D-glucosidase activity of Oenococcus SD-2a, Food Science and Technology, 2022, 38(08): 48-52.

[0063] The method for determining aroma in the following examples is as follows: refer to Lan Rong et al., Effect of glucose oxidase on aroma components of blueberry wine during storage, 2015, 36(7): 110-114.

[0064] Example 1: Wine fermentation culture

[0065] (1) Activation of strains: Streaking of frozen Oenococcus oeni VP41 (purchased from Shanghai Jietu Biotechnology Co., Ltd.) on solid culture medium was activated and cultured at 20°C for 15 days. The solid culture medium contained 5 g / L glucose, 150 mL / L Jerusalem artichoke pulp, 5 g / L yeast extract powder, 8 g / L peptone, 0.15 g / L MgSO4·7H2O, 0.02 g / L MnSO4·4H2O, pH value was adjusted to 4.3, and 20 g / L agar was added. 800 g washed Jerusalem artichoke was chopped, mixed evenly with 200 g purified water, and then beaten to obtain Jerusalem artichoke pulp.

[0066] (2) Expansion culture of strains: Pick a single colony on the solid culture medium, inoculate it into 500 mL of liquid culture medium, and culture it at 20°C for 10 days. The liquid culture medium contains 8 g / L of cellobiose, 150 mL / L of Jerusalem artichoke pulp, 5 g / L of yeast extract powder, 8 g / L of peptone, 0.15 g / L of MgSO4·7H2O, and 0.02 g / L of MnSO4·4H2O, and the pH value is adjusted to 4.3.

[0067] (3) Induction culture of bacterial strains: 500 mL of the expanded culture medium was centrifuged (5000 g, 5 min) to obtain the Oenococcus oeni VP41 bacterial cells, which were then added to 500 mL of induction liquid culture medium A and cultured at 18°C ​​for 3 days. On the second day, the cells were subjected to ultrasonic treatment (50 w, 20 min). 500 mL of the culture medium was centrifuged again (5000 g, 5 min) to obtain the bacterial cells, which were added to 500 mL of induction liquid culture medium B and cultured at 18°C ​​for 4 days. On the second day, the cells were subjected to ultrasonic treatment (50 w, 20 min).

[0068] The carbon source in the induction liquid medium A is a mixture of Jerusalem artichoke extract and arbutin, the weight ratio of the two is 1:5, the addition amount is 11g / L, and the other components are yeast extract powder 5g / L, peptone 8g / L, MgSO4·7H2O 0.15g / L, MnSO4·4H2O 0.02g / L, pH 4.3; the carbon source in the induction liquid medium B is a mixture of Jerusalem artichoke extract and two disaccharide glycosides (genipin gentian disaccharide and stevioside, the mass ratio of the two is 1:1), the mass ratio of the mixture of Jerusalem artichoke extract and disaccharide glycoside is 1:7, the addition amount is 12g / L, and the other components are yeast extract powder 5g / L, peptone 8g / L, MgSO4·7H2O 0.15g / L, MnSO4·4H2O0.02g / L, pH 4.3.

[0069] (4) Adaptation culture of strains: 500 mL of the secondary induction culture fluid was centrifuged (5000 g, 5 min) to obtain bacterial cells, which were added to 500 mL of adaptation medium and cultured at 18°C ​​for 5 days. The adaptation medium contained 5 g / L glucose, 5% (v / v) ethanol, 20 mg / L SO2, 15 mL / L glucosidase extract, 3 g / L tartaric acid, 1 g / L malic acid, 1 g / L K2HPO4, 0.1 g / L MgSO4·7H2O, 0.02 g / L MnSO4·H2O, 1 g / L yeast extract, and the pH was adjusted to 3.5. Finally, the medium was filtered with a 0.22 μm filter membrane.

[0070] (5) Fermentation: The adapted culture solution was centrifuged (5000 g, 5 min) to obtain the bacterial cells, which were inoculated at an inoculum size of 8 g / L into 1 L of wine with a total sugar content of 12 g / L and a SO2 content of 22 mg / L at 18°C ​​for 20 days. The wine was then ultrasonically treated at 100 W for 30 min on the 5th and 10th days, respectively.

[0071] The bacterial glycosidase activity and fruit wine aroma of the wine obtained above were determined, and the results are shown in Tables 1 and 2.

[0072] Table 1 Results of determination of glycosidase activity of Oenococcus oeni in wine

[0073]

[0074] Table 2 Determination results of fruit wine aroma in wine

[0075] Aroma Type Relative content Esters Alcohols Aldehydes and Ketones Terpenes Alkanes other 56 89.35％ 18 12 11 9 5 1

[0076] In summary, by adding carbon source glucose and Jerusalem artichoke pulp in the activation and expansion steps of Oenococcus oeni VP41 strain, adding carbon source Jerusalem artichoke extract and arbutin monosaccharide in the first induction culture in the strain induction culture step, adding carbon source Jerusalem artichoke extract, genipin gentianobioside and stevioside in the second induction culture, using ultrasonic treatment under different conditions in strain induction culture and fermentation culture, and finally adjusting fermentation parameters in fermentation culture, the various glycosidase activities of the obtained bacteria in the wine are high, and the obtained wine has many types and high contents of aroma substances. Through sensory evaluation, the aroma is fresh and fruity.

[0077] Example 2: Fermentation of blueberry wine

[0078] (1) Activation of strains: Streaking of frozen Oenococcus oeni 31MBR (purchased from Shanghai Jietu Biotechnology Co., Ltd.) on solid culture medium was activated and cultured at 25°C for 10 days. The solid culture medium contained 8 g / L glucose, 100 mL / L Jerusalem artichoke pulp, 5 g / L yeast extract powder, 8 g / L peptone, 0.15 g / L MgSO4·7H2O, 0.02 g / L MnSO4·4H2O, pH value was adjusted to 4.5, and 20 g / L agar was added. 800 g washed Jerusalem artichoke was chopped, mixed evenly with 200 g purified water, and then beaten to obtain Jerusalem artichoke pulp.

[0079] (2) Expansion culture of strains: Pick a single colony on the solid culture medium, inoculate it into 2L of liquid culture medium, and culture it at 25°C for 7 days. The liquid culture medium contains 10g / L cellobiose, 100mL / L Jerusalem artichoke pulp, 5g / L yeast extract powder, 8g / L peptone, 0.15g / L MgSO4·7H2O, 0.02g / L MnSO4·4H2O, and the pH value is adjusted to 4.5.

[0080] (3) Induction culture of bacterial strains: 2 L of expanded culture medium was centrifuged (5000 g, 5 min) to obtain Oenococcus oeni 31MBR cells, which were then added to 2 L of induction liquid culture medium A and cultured at 20°C for 2 days. On the second day, ultrasonic treatment was performed (100 w, 15 min); 2 L of culture medium was centrifuged again (5000 g, 5 min) to obtain cells, which were added to 2 L of induction liquid culture medium B and cultured at 20°C for 3 days. On the second day, ultrasonic treatment was performed again (100 w, 15 min).

[0081] The carbon source in the induction liquid medium A is a mixture of Jerusalem artichoke extract and raspberry glycosides, the weight ratio of the two is 1:6, the addition amount is 12g / L, other ingredients are yeast extract 5g / L, peptone 8g / L, MgSO4·7H2O 0.15g / L, MnSO4·4H2O0.02g / L, pH 4.5; the carbon source in the induction liquid medium B is a mixture of Jerusalem artichoke extract and two disaccharide glycosides (schizonepeta tenuifolia disaccharide and gentiana gentiana disaccharide, the mass ratio of the two is 1:1), the mass ratio of the mixture of Jerusalem artichoke extract and disaccharide glycoside is 1:8, the addition amount is 14g / L, other ingredients are yeast extract 5g / L, peptone 8g / L, MgSO4·7H2O 0.15g / L, MnSO4·4H2O 0.02g / L, pH 4.5.

[0082] (4) Adaptation culture of strains: 2L of secondary induction culture fluid was centrifuged (5000g, 5min) to obtain bacterial cells, which were added to 2L of adaptation medium and cultured at 20°C for 4 days. The adaptation medium contained 8g / L glucose, 8% (v / v) ethanol, 25mg / L SO2, 20mL / L glucosidase extract, 4g / L tartaric acid, 2g / L malic acid, 1.5g / L K2HPO4, 0.15g / L MgSO4·7H2O, 0.04g / L MnSO4·H2O, 1.8g / L yeast extract, and the pH was adjusted to 3.8. Finally, the medium was filtered with a 0.22μm filter membrane.

[0083] (5) Fermentation culture: The adapted culture solution was centrifuged (5000 g, 5 min) to obtain the bacterial cells, which were inoculated at an inoculum size of 10 g / L into 2 L of blueberry wine with a total sugar content of 19 g / L and a SO2 content of 29 mg / L at 20°C for 15 days, and ultrasonically treated at 150 W for 20 min on the 5th and 10th days, respectively.

[0084] The blueberry wine obtained above was subjected to bacterial glycosidase activity assay and wine aroma assay, and the results are shown in Tables 3 and 4.

[0085] Table 3 Determination results of glycosidase activity of Oenococcus oeni in blueberry wine

[0086]

[0087] Table 4 Determination results of fruit wine aroma in blueberry wine

[0088] Aroma Type Relative content Esters Alcohols Aldehydes and Ketones Terpenes Alkanes other 58 90.35 21 9 10 10 6 2

[0089] In summary, by adding carbon source glucose and Jerusalem artichoke pulp in the activation and expansion culture steps of the wine Oenococcus oeni 31MBR strain, adding carbon source Jerusalem artichoke extract and raspberry glycoside monosaccharide in the first induction culture in the strain induction culture step, adding carbon source Jerusalem artichoke extract, scutellaria disaccharide and genipin gentiobioside in the second induction culture, using ultrasonic treatment under different conditions in the strain induction culture and fermentation culture, and finally adjusting the fermentation parameters in the fermentation culture, the various glycosidase activities of the obtained bacteria in the blueberry wine are all high, and the obtained wine has many types and high contents of aroma substances. The obtained blueberry wine was sensory evaluated, and the aroma was fresh, rich in fruity aroma, and had the typical aroma of blueberries.

[0090] Comparative Example 1: Effect of Jerusalem artichoke pulp / Jerusalem artichoke extract without adding carbon source on the effect of the invention

[0091] In steps (1), (2) and (3), the Jerusalem artichoke pulp / Jerusalem artichoke extract was not added as the carbon source to culture Oenococcus oeni VP41, and an equal amount of glucose was used to replace the Jerusalem artichoke pulp / Jerusalem artichoke extract. The other steps and parameters were the same as those in Example 1, and the bacterial glycosidase activity and fruit wine aroma were detected when the carbon source Jerusalem artichoke pulp / Jerusalem artichoke extract was not added.

[0092] The results are shown in Table 5 and Table 6:

[0093] Table 5 Glycosidase activity of bacteria without adding Jerusalem artichoke pulp / Jerusalem artichoke extract

[0094]

[0095] Table 6 Effect of not adding Jerusalem artichoke pulp / Jerusalem artichoke extract on the aroma of fruit wine

[0096] Aroma Type Relative content Esters Alcohols Aldehydes and Ketones Terpenes Alkanes other 46 73.98％ 16 9 8 8 3 2

[0097] As shown in the results of Tables 5 and 6, when Jerusalem artichoke pulp / Jerusalem artichoke extract is not added in steps (1), (2) and (3), the various glycosidase activities of the obtained bacteria are significantly different from those in Example 1, the glycosidase activity is not high, and the types and contents of wine aroma substances are not high either.

[0098] Comparative Example 2: Effect of not performing ultrasonic treatment in the induction culture step on the effect of the present invention

[0099] No ultrasonic treatment was performed in the induction culture step. The other steps and parameters were the same as those in Example 2. Oenococcus oeni 31MBR was treated, and then the bacterial glycosidase activity and fruit wine aroma were detected when no ultrasonic treatment was performed in the induction culture step.

[0100] The results are shown in Tables 7 and 8:

[0101] Table 7 Glycosidase activity of bacteria without ultrasonic treatment in the induction culture step

[0102]

[0103] Table 8 Effect of no ultrasonic treatment on the aroma of fruit wine in the induction culture step

[0104] Aroma Type Relative content Esters Alcohols Aldehydes and Ketones Terpenes Alkanes other 44 76.32 15 10 7 6 3 3

[0105] As shown in the results of Tables 7 and 8, when ultrasonic treatment is not performed in the induction culture step, the various glycosidase activities, types and contents of wine aroma substances in the obtained bacteria are not higher than those in Example 2.

[0106] Comparative Example 3: Effect of not adding carbon source monosaccharide / disaccharide to the induction medium on the effect of the present invention

[0107] In the induction culture step, no carbon source monosaccharide / disaccharide was added, and an equal amount of glucose was used instead of the carbon source monosaccharide / disaccharide. The other steps and parameters were the same as in Example 1. Oenococcus oeni VP41 was treated, and the bacterial glycosidase activity and fruit wine aroma were detected when no carbon source monosaccharide / disaccharide was added in the induction culture step.

[0108] The results are shown in Tables 9 and 10:

[0109] Table 9 Glycosidase activity of bacteria when no carbon source monosaccharide / disaccharide was added to the induction medium

[0110]

[0111] Table 10 Effect of not adding carbon source monosaccharide / disaccharide to the induction medium on the aroma of fruit wine

[0112] Aroma Type Relative content Esters Alcohols Aldehydes and Ketones Terpenes Alkanes other 47 70.24 16 9 9 7 4 2

[0113] As shown in the results of Tables 9 and 10, when no carbon source monosaccharide / disaccharide was added to the induction medium, compared with Example 1, the activities of various glycosidases in the obtained bacteria were not high, and the types and contents of aroma substances in the obtained wine were also not high.

[0114] The present invention provides a method and idea for increasing the glycosidase activity of intact cells of brewing lactic acid bacteria during fermentation. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for increasing the glycosidase activity of intact cells of brewing lactic acid bacteria during fermentation, characterized in that: The steps include: (1) Activation of strains: streaking the frozen lactic acid bacteria on a solid culture medium and culturing at 20-25°C for 10-15 days, wherein the carbon source of the solid culture medium is 5-8 g / L glucose and 100-150 mL / L Jerusalem artichoke pulp; (2) Expanding the culture of the strain: picking a single colony after streaking culture in step (1), and expanding the culture in a liquid culture medium at 20-25° C. for 7-10 days; the carbon source of the liquid culture medium is: 8-10 g / L cellobiose, 100-150 mL / L Jerusalem artichoke pulp; (3) Induction culture of bacterial strains: the culture fluid obtained after the expanded culture in step (2) is centrifuged at 5000 g for 5 min, and the bacterial bodies are inoculated into induction liquid culture medium A for primary induction culture; the bacterial bodies obtained by centrifuging the primary induction culture fluid again at 5000 g for 5 min are inoculated into induction liquid culture medium B for secondary induction culture; ultrasonic treatment is performed at 50-100 W for 15-20 min on the second day of the primary induction culture and the second induction culture respectively; the carbon source of the induction liquid culture medium A is a mixture of Jerusalem artichoke extract and monosaccharide glycosides; the carbon source of the induction liquid culture medium B is a mixture of Jerusalem artichoke extract and disaccharide glycosides; (4) Adaptation culture of bacterial strains: The bacterial cells obtained by centrifuging the secondary induction culture solution in step (3) at 5000 g for 5 min were inoculated into the adaptation medium and cultured at 18-20°C for 4-5 days; (5) Fermentation culture: the adapted culture solution obtained after the adaptation culture in step (4) is centrifuged at 5000 g for 5 min, and the cells are inoculated into a fruit wine having a total sugar content of 10-20 g / L and a SO2 content of less than 30 mg / L at an inoculum amount of 8-10 g / L of wet cells, and fermented at 18-20° C. for 15-20 days, and ultrasonically treated at 100-150 W for 20-30 min on the 5th and 10th days of fermentation culture; Wherein, in step (1), the brewing lactic acid bacteria is Oenococcus oeni Oenococcus oeni ; Wherein, in step (1) and step (2), the Jerusalem artichoke pulp is prepared by mixing the washed Jerusalem artichoke and purified water in a weight ratio of 8:2 and beating the mixture; Wherein, in step (3), the culture conditions of the first induction culture are: 18-20°C, culture for 2-3 days, and ultrasonic treatment on the second day, the treatment conditions are 50-100 w, and the ultrasonic time is 15-20 min; the culture conditions of the second induction are 18-20°C, culture for 3-4 days, and ultrasonic treatment on the second day, the treatment conditions are 50-100 w, and the ultrasonic time is 15-20 min; the carbon source of the induction liquid culture medium A is a mixture of Jerusalem artichoke extract and monosaccharide, the weight ratio of the two is 1:5-1:6, the addition amount is 11-12 g / L, and the other ingredients are yeast extract powder 5 g / L, peptone 8 g / L, MgSO4•7H2O0.15 g / L, MnSO4•4H2O 0.02 g / L, pH 4.3-4.5; the carbon source of the induction liquid medium B is a mixture of Jerusalem artichoke extract and disaccharide, the weight ratio of the two is 1:7-1:8, the addition amount is 12-14 g / L, the other ingredients are yeast extract powder 5 g / L, peptone 8 g / L, MgSO4•7H2O 0.15 g / L, MnSO4•4H2O 0.02 g / L, pH 4.3-4.5; Wherein, in step (3), the Jerusalem artichoke extract is extracted by the following method: fresh Jerusalem artichoke is washed, sliced, soaked, dried and crushed to obtain Jerusalem artichoke powder, the Jerusalem artichoke powder is mixed with water at a weight ratio of 1:10, ultrasonically extracted at 70-80°C for 30min-45min, rotary evaporated at 35°C and allowed to stand at 0-4°C to obtain crystals, and dried to obtain the Jerusalem artichoke extract; The monosaccharide glycoside is any one of arbutin and raspberry glycoside, and the disaccharide glycoside is a combination of any two of genipin gentianobioside, stevia bioside, and ophiopogon disaccharide, and the mass ratio thereof is 1:

1.

2. The method according to claim 1, characterized in that: In step (1), the solid culture medium has a carbon source of 5-8 g / L glucose, 100-150 mL / L Jerusalem artichoke pulp, and other ingredients of 5 g / L yeast extract, 8 g / L peptone, 0.15 g / L MgSO4•7H2O, 0.02 g / L MnSO4•4H2O, pH 4.3-4.5, and 20 g / L agar.

3. The method according to claim 1, characterized in that In step (2), the liquid culture medium comprises 8-10 g / L of cellobiose, 100-150 mL / L of Jerusalem artichoke pulp, 5 g / L of yeast extract powder, 8 g / L of peptone, 0.15 g / L of MgSO4•7H2O, 0.02 g / L of MnSO4•4H2O, and a pH of 4.3-4.

5.

4. The method according to claim 1, characterized in that: In step (4), the adaptation medium comprises the following components: glucose 5-8 g / L, ethanol content 5-8% v / v, SO2 content 20-25 mg / L, glucosidase extract 15-20 mL / L, tartaric acid 3-4 g / L, malic acid 1-2 g / L, K2HPO4 1-1.5 g / L, MgSO4•7H2O 0.1-0.15 g / L, MnSO4•H2O 0.02-0.04 g / L, yeast extract 1-1.8 g / L, and pH 3.5-3.

8.

5. The method according to claim 1, characterized in that In step (5), the fruit wine mainly includes any one of grape wine, blueberry wine, apple wine and crabapple wine.

6. The method according to claim 1, characterized in that The glycosidase includes glucosidase, arabinosidase, rhamnosidase and apiosidase.

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