CN111919987B - Method for simultaneously promoting malic acid lactic acid conversion and polyphenol derivation by ultrasonic-assisted lactobacillus plantarum fermented apple juice - Google Patents
Method for simultaneously promoting malic acid lactic acid conversion and polyphenol derivation by ultrasonic-assisted lactobacillus plantarum fermented apple juice Download PDFInfo
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Abstract
The invention discloses a method for simultaneously promoting malic acid-lactic acid conversion and polyphenol derivatization by ultrasonic-assisted lactobacillus plantarum fermented apple juice, and relates to the technical field of food processing of lactobacillus fermented fruit juice. The method utilizes ultrasonic waves to perform auxiliary treatment on the process of fermenting the apple juice by lactobacillus plantarum, and regulates and controls the process of fermenting the apple juice by regulating parameter indexes such as ultrasonic intervention period, power density and the like. According to the invention, delay-phase ultrasonic treatment and logarithmic-phase ultrasonic treatment can accelerate lactobacillus plantarum proliferation and malic acid-lactic acid conversion in the fermentation process, and promote polyphenol derivation in apple juice, including hydrolysis of chlorogenic acid to generate caffeic acid, procyanidine decomposition, catechin enrichment and gallic acid decarboxylation, so that the antioxidant activity of the fermented apple juice is improved, but the stationary-phase ultrasonic treatment has no obvious influence. The invention has great economic benefit and social benefit and good application prospect.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to a method for assisting lactobacillus plantarum in fermenting apple juice and promoting lactic acid conversion of malic acid and polyphenol derivatization simultaneously through ultrasonic treatment.
Technical Field
The apple is rich in various nutritional ingredients such as organic acid, dietary fiber, vitamins and polyphenol. The apple can be used for inhibiting cardiovascular diseases and diabetes. China is the world with the largest apple yield and the largest consumer country internationally, and the annual yield reaches 4300 ten thousand tons. However, in the apple industry of China, 64 percent of apples are used for fresh fruits, 25 percent of apples are used for concentrated juice processing, other products only account for 10 percent, and the apples are deeply processedThe mode is deficient. In recent years, probiotic fermented fruit and vegetable juice is receiving wide attention. However, probiotics, especially lactobacillus plantarum, is highly appreciated by consumers due to its outstanding effects in regulating intestinal flora, enhancing immunity, relieving gastrointestinal diseases, improving health, etc. Therefore, the fermented apple juice rich in lactobacillus plantarum has better production prospect and wide consumption market. Researches show that malic acid and lactic acid conversion exists in the fermentation process of lactobacillus plantarum, namely, lactobacillus takes L-malic acid with double carboxyl as a substrate and is converted into L-lactic acid with single carboxyl and CO under the catalysis of malic acid lactase 2 The conversion process enables the lactobacillus plantarum to proliferate by taking organic acid as a main carbon source, breaks through the technical bottleneck of lactic acid fermentation, and endows the fruit and vegetable juice with pleasant and harmonious fermentation fragrance; in addition, flavor derivatives generated by degradation of astringent phenolic substances and reduction of phenolic acids such as caffeic acid can also improve the flavor and mouthfeel of apple juice. Meanwhile, in the lactic acid fermentation process, polyphenol derivatization can generate substances with stronger antioxidant activity through decarboxylation, reduction and other reactions, so that the antioxidant activity of the apple juice is enhanced, and the nutritional value of the apple juice is improved. However, the apple juice has high acidity, and the system lacks growth factors required by lactobacillus plantarum fermentation, so that the defects of slow microbial growth, incapability of quickly forming dominant flora and easy contamination by infectious microbes, low conversion rate of phenolic substances and the like exist in the production of lactobacillus plantarum fermented apple juice.
Patent 201811074098.X discloses a method for preparing fermented apple juice rich in active lactobacillus plantarum by adding proliferation factors and inorganic salts for promoting lactobacillus plantarum proliferation to the fruit juice, so that lactobacillus plantarum can be rapidly fermented in apple juice. Patent 202010243654.2 discloses a method for improving bioavailability of polyphenol in apple juice, which adopts rhamnose bacillus, and after activation and a series of domestication, apple juice is fermented, so that apple polyphenol is converted into a substance which is easy to absorb by small molecules, and thus bioavailability of polyphenol in vivo is improved. Naturally, the proliferation factor is added to enable the lactobacillus plantarum to be rapidly fermented in the apple juice, and the viable count reaches 10 9 CFU/mL, but in the method of patent 202010243654.2, the earlier strain had been acclimatizedThe process generally consumes about 7 days, which is not favorable for industrial high-efficiency production. Therefore, it is necessary to develop a simpler and more efficient technique for enhancing the polyphenol conversion of apple juice in lactic acid fermentation, improving the bioavailability of polyphenol, and further accelerating the microbial growth and malic acid lactic acid conversion based on the prior art.
Relevant studies have shown that moderate ultrasound stimulation can alter the permeability of cell membranes and enhance substance transport. The ultrasonic treatment can not only promote the growth and proliferation of microorganisms, enhance the metabolic reaction activity and improve the yield of fermentation products, but also promote the conversion of bioactive substances and improve the bioactivity of fermentation liquor. Thus, the ultrasonic wave can be applied to the development of functional fermented products. Patent CN103865714 discloses a method for brewing yellow wine with the assistance of low-intensity intermittent ultrasonic waves, which utilizes the low-intensity intermittent ultrasonic waves to perform auxiliary treatment on the yellow wine brewing process, regulates and controls the yellow wine fermentation process by adjusting the ultrasonic frequency, power, working time, intermittent time and the like, and consequently greatly shortens the yellow wine fermentation time, and all indexes of the yellow wine brewed with the assistance of the ultrasonic waves and the yellow wine brewed without the ultrasonic waves are basically consistent. Patent 201610208464.0 discloses that in the fermentation process of lactic acid bacteria, the biomass of lactic acid bacteria is increased by 12-55% and the acid production is increased by 15-60% by using ultrasound as an auxiliary means. However, the existing patents do not show the influence of ultrasonic treatment on the growth and related metabolism of microorganisms in different growth periods, and articles and patents for enhancing polyphenol derivatization and malic acid-lactic acid conversion in the process of fermenting fruit and vegetable juice by using ultrasonic treatment are rarely reported so far.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a method for promoting malic acid-lactic acid conversion and polyphenol derivatization by fermenting apple juice with lactobacillus plantarum under the assistance of ultrasonic waves. According to the invention, on the basis of adding the proliferation factor, the proliferation rate of lactobacillus plantarum in the fermentation process is further improved, malic acid-lactic acid conversion and polyphenol derivatization are promoted, and the antioxidant activity of the fermented apple juice is improved; at the same time, it is clear that these beneficial effects are achieved only in the lag phase and log phase of microbial growth, whereas stationary phase sonication cannot. The method has the characteristics of simple process, advanced technology, high safety, low production cost, suitability for industrial production and the like.
The technical scheme adopted by the invention for solving the technical problem is as follows:
a method for assisting Lactobacillus plantarum in fermenting apple juice and promoting malic acid lactic acid conversion and polyphenol derivatization by ultrasonic treatment comprises the following specific steps of taking Lactobacillus plantarum (Lactobacillus plantarum) as a starting strain:
(1) Preparing a seed culture medium and apple juice;
(2) Inoculating lactobacillus plantarum to a seed culture medium activating strain;
(3) Inoculating the activated strain into apple juice, and measuring the growth curve;
(4) Inoculating the activated strain into apple juice, performing ultrasonic treatment, and continuously culturing the treated apple juice to obtain a fermented apple juice product.
As a preferred technical scheme of the application, the main component of the seed culture medium in the step (1) is MRS broth, and the preparation method comprises the following steps: commercially available MRS broth 49.3g was weighed, dissolved in 1L water and autoclaved at 121 ℃ for 20min.
As a preferred technical scheme of the application, the preparation method of the apple juice in the step (1) comprises the following steps: selecting a mature red Fuji apple with excellent properties as a raw material, cleaning and airing, removing peel and fruit core, cutting, crushing apple blocks by using a beater, adding 0.1-0.5% of D-sodium erythorbate and 10% of purified water while crushing, filtering obtained apple pulp by using gauze to remove precipitates, centrifuging apple juice, taking supernatant to obtain apple juice, adjusting the pH value to 6.0, adding 0.1-0.8% of yeast extract powder, and sterilizing for later use.
Preferably, the gauze has 120 meshes.
Preferably, the centrifugation conditions are: centrifuging at 4000r/min for 20min.
Preferably, the sterilization conditions are: sterilizing at 85 deg.C for 20min.
As a preferred embodiment of the present invention, the method for measuring the growth curve in step (3) comprises: selecting Lactobacillus plantarum, inoculating into 50mL sterile MRS broth, and culturing at 37 deg.C for 24h; inoculating the cultured bacterial liquid into apple juice according to the inoculation amount of 2%, controlling the temperature to be 30 ℃ by adopting a temperature-controlled bacterial growth tester, and periodically testing the light absorption value of the apple juice at 600 nm; and drawing a growth curve by taking the light absorption value as the y axis and the culture time as the x axis.
As a preferred technical scheme of the application, the steps and conditions for inoculating the activated strains in the step (3) and the step (4) into the apple juice are as follows: the seed liquid was inoculated into apple juice at an inoculum size of 2% with an initial bacterial count of about 7.0Log CFU/mL and fermented at 30 ℃.
As a preferred embodiment of the present invention, the ultrasonic treatment method and conditions in the step (4) are as follows: according to the measuring result of the growth curve, ultrasonic treatment is carried out in the stationary phase (0-4 h after fermentation) or logarithmic phase (4-12 h after fermentation) of the growth of the lactobacillus plantarum in the apple juice, the ultrasonic power density is 10-100W/L, the ultrasonic frequency is 20-30 kHz, the ultrasonic time is 10-60 min, and the ultrasonic pulse sounding condition is 5-20s of ultrasonic pause for 5-20s.
As a preferred technical scheme of the application, the method and conditions for continuously culturing the apple juice in the step (4) are as follows: and placing the sample after ultrasonic treatment in an incubator at 30 ℃ for static culture for 24 hours.
Advantageous effects
Compared with the prior art, the invention has the following advantages:
(1) Further promoting the thallus multiplication and malic acid and lactic acid conversion in the process of fermenting apple juice by lactobacillus plantarum. The ultrasonic wave increases the number of viable bacteria in the apple juice by 30-200% in the fermentation process compared with the ultrasonic-free group, the lactic acid content is increased by 5-70% in the fermentation process compared with the ultrasonic-free group, and the malic acid content is reduced by 5-40% in the fermentation process compared with the ultrasonic-free group. The rapid increase of the number of the lactobacillus plantarum and the lactic acid content enables the formation of dominant flora and acidic fermentation environment to be rapid, and the risk of mixed bacteria pollution is reduced.
(2) The method directly enhances the polyphenol derivatization in the fermentation process of the apple juice by low-intensity ultrasonic treatment, improves the antioxidant activity of the apple juice by 5-30 percent in comparison with that of the apple juice without an ultrasonic group in the fermentation process, omits a strain domestication process, greatly shortens the production period and is beneficial to industrial high-efficiency production.
(3) The present invention is clear that these beneficial effects can be achieved only in the lag phase and logarithmic phase of microbial growth, whereas stationary phase sonication cannot.
(4) The method has the characteristics of simple process, advanced technology, high safety, low production cost, suitability for industrial production and the like.
Drawings
FIGS. 1a, b, c show the comparison of viable bacteria during fermentation of example 1 and comparative example 1, example 2 and comparative example 1, and comparative example 2 and comparative example 1, respectively;
FIG. 2,3,4 is a comparison of in vitro antioxidant activity during fermentation for example 2 and comparative example 1, example 1 and comparative example 1, and comparative example 2 and comparative example 1, respectively.
Detailed Description
The present invention is further illustrated by the following examples.
The Lactobacillus plantarum (Lactobacillus plantarum) used in the specific embodiment of the invention is Lactobacillus plantarum BNCC337796 lyophilized powder, purchased from beijing beina biological collection center.
Example 1
(1) Preparing a seed culture medium and apple juice:
weighing 49.3g of commercially available MRS broth, dissolving in 1L of water, autoclaving at 121 deg.C for 20min, and cooling to obtain seed culture medium;
selecting mature red Fuji apples with excellent properties as a raw material, cleaning and airing, removing peels and fruit cores, cutting, crushing apple blocks by using a beater, adding 0.15% of D-sodium erythorbate and 10% of purified water while crushing, filtering obtained apple pulp by using 120-mesh gauze to remove fruit residues, then centrifuging apple juice at 4000r/min for 20min, taking supernatant to obtain apple juice, adjusting the pH to 6.0, adding 0.3% of yeast extract powder, and sterilizing at 85 ℃ for 20min for later use.
(2) Inoculating lactobacillus plantarum in a seed culture medium to activate strains:
lactobacillus plantarum was picked and inoculated into 50mL sterile MRS broth and cultured at 37 ℃ for 24h.
(3) Inoculating the activated strain into apple juice, and measuring the growth curve:
inoculating the cultured bacterial liquid into apple juice according to the inoculation amount of 2%, controlling the temperature to be 30 ℃ by adopting a temperature-controlled bacterial growth tester, and regularly measuring the light absorption value of the apple juice at 600 nm. And drawing a growth curve by taking the light absorption value as the y axis and the culture time as the x axis.
(4) Ultrasonic-assisted fermentation of apple juice:
inoculating the activated strain into apple juice according to the inoculation amount of 2%, wherein the initial colony number of the apple juice fermentation is about 7.0Log CFU/mL, and fermenting at 30 ℃. And (3) carrying out ultrasonic treatment after lactobacillus plantarum is fermented for 0h (in a lag phase), wherein the ultrasonic power density is 93.6W/L, the ultrasonic frequency is 20kHz, the ultrasonic time is 30min, and the ultrasonic pulse sounding condition is 5s of ultrasonic and 5s of ultrasonic interval. After the ultrasonic treatment, the sample is placed in an incubator at 30 ℃ for static culture for 24 hours.
Example 2
(1) Preparing a seed culture medium and apple juice:
weighing 49.3g of commercially available MRS broth, dissolving in 1L of water, autoclaving at 121 deg.C for 20min, and cooling to obtain seed culture medium;
selecting mature red Fuji apples with excellent properties as a raw material, cleaning and airing, removing peels and fruit cores, cutting, crushing apple blocks by using a beater, adding 0.15% of D-sodium erythorbate and 10% of purified water while crushing, filtering obtained apple pulp by using 120-mesh gauze to remove fruit residues, then centrifuging apple juice at 4000r/min for 20min, taking supernatant to obtain apple juice, adjusting the pH to 6.0, adding 0.3% of yeast extract powder, and sterilizing at 85 ℃ for 20min for later use.
(2) Inoculating lactobacillus plantarum to a seed culture medium to activate strains:
lactobacillus plantarum was picked and inoculated into 50mL sterile MRS broth and cultured at 37 ℃ for 24h.
(3) Inoculating the activated strain into apple juice, and measuring the growth curve:
inoculating the cultured bacterial liquid into apple juice according to the inoculation amount of 2%, controlling the temperature to be 30 ℃ by adopting a temperature-controlled bacterial growth tester, and regularly measuring the light absorption value of the apple juice at 600 nm. And drawing a growth curve by taking the light absorption value as the y axis and the culture time as the x axis.
(4) Ultrasonic-assisted fermentation of apple juice:
inoculating the activated strain into apple juice according to the inoculation amount of 2%, wherein the initial colony number of the apple juice fermentation is about 7.0Log CFU/mL, and fermenting at 30 ℃. After lactobacillus plantarum is fermented for 8 hours (in a logarithmic phase), ultrasonic treatment is carried out, the ultrasonic power density is 93.6W/L, the ultrasonic frequency is 20kHz, the ultrasonic time is 30min, and the ultrasonic pulse sounding condition is 5s of ultrasonic interval and 5s of ultrasonic pulse sounding. After the ultrasonic treatment, the sample is placed in an incubator at 30 ℃ for static culture for 24 hours.
Comparative example 1
The same fermentation as in examples 1 and 2 was conducted except that the ultrasonic treatment was not conducted.
Comparative example 2
The conditions were the same as in examples 1 and 2 except that sonication was carried out 18h after fermentation of the apple juice (stationary phase).
Example 3
(1) Preparing a seed culture medium and apple juice:
weighing 49.3g of commercially available MRS broth, dissolving in 1L of water, autoclaving at 121 deg.C for 20min, and cooling to obtain seed culture medium;
selecting mature red Fuji apples with excellent properties as a raw material, cleaning and airing, removing peels and fruit cores, cutting, crushing apple blocks by using a beater, adding 0.1% of D-sodium erythorbate and 10% of purified water while crushing, filtering obtained apple pulp by using 120-mesh gauze to remove fruit residues, then centrifuging apple juice at 4000r/min for 20min, taking supernatant to obtain apple juice, adjusting the pH to 6.0, adding 0.8% of yeast extract powder, and sterilizing at 85 ℃ for 20min for later use.
(2) Inoculating lactobacillus plantarum to a seed culture medium to activate strains:
lactobacillus plantarum was picked and inoculated into 50mL sterile MRS broth and cultured at 37 ℃ for 24h.
(3) Inoculating the activated strain into apple juice, and measuring the growth curve:
inoculating the cultured bacterial liquid into apple juice according to the inoculation amount of 2%, controlling the temperature to be 30 ℃ by adopting a temperature-controlled bacterial growth tester, and regularly measuring the light absorption value of the apple juice at 600 nm. And drawing a growth curve by taking the light absorption value as the y axis and the culture time as the x axis.
(4) Ultrasonic-assisted fermentation of apple juice:
inoculating the activated strain into apple juice according to the inoculation amount of 2%, wherein the initial colony number of the apple juice fermentation is about 7.0Log CFU/mL, and fermenting at 30 ℃. After lactobacillus plantarum is fermented for 6 hours (logarithmic phase), ultrasonic treatment is carried out, the ultrasonic power density is 100W/L, the ultrasonic frequency is 20kHz, the ultrasonic time is 10min, and the ultrasonic pulse sounding condition is ultrasonic 5s and interval 20s. After the ultrasonic treatment, the sample is placed in an incubator at 30 ℃ for static culture for 24 hours.
Example 4
(1) Preparing a seed culture medium and apple juice:
weighing 49.3g of commercially available MRS broth, dissolving in 1L of water, autoclaving at 121 deg.C for 20min, and cooling to obtain seed culture medium;
selecting mature red Fuji apples with excellent properties as a raw material, cleaning and airing, removing peels and fruit cores, cutting, crushing apple blocks by using a beater, adding 0.5% of D-sodium erythorbate and 10% of purified water while crushing, filtering obtained apple pulp by using 120-mesh gauze to remove fruit residues, then centrifuging apple juice at 4000r/min for 20min, taking supernatant to obtain apple juice, adjusting the pH to 6.0, adding 0.1% of yeast extract powder, and sterilizing at 85 ℃ for 20min for later use.
(2) Inoculating lactobacillus plantarum in a seed culture medium to activate strains:
lactobacillus plantarum was picked and inoculated in 50mL sterile MRS broth and incubated at 37 ℃ for 24h.
(3) Inoculating the activated strain into apple juice, and measuring the growth curve:
inoculating the cultured bacterial liquid into apple juice according to the inoculation amount of 2%, controlling the temperature to be 30 ℃ by adopting a temperature-controlled bacterial growth tester, and regularly measuring the light absorption value of the apple juice at 600 nm. And drawing a growth curve by taking the light absorption value as the y axis and the culture time as the x axis.
(4) Ultrasonic-assisted fermentation of apple juice:
inoculating the activated strain into apple juice according to the inoculation amount of 2%, wherein the initial colony number of the apple juice fermentation is about 7.0Log CFU/mL, and fermenting at 30 ℃. And (3) carrying out ultrasonic treatment after lactobacillus plantarum is fermented for 2 hours (in a lag phase), wherein the ultrasonic power density is 10W/L, the ultrasonic frequency is 30kHz, the ultrasonic time is 60min, and the ultrasonic pulse sounding condition is that the ultrasonic pulse sounding condition is 20s and the ultrasonic interval is 5s. After the ultrasonic treatment, the sample is placed in an incubator at 30 ℃ for static culture for 24 hours.
Influence of ultrasound on microbial growth in apple juice fermentation process in different growth periods
As shown in FIG. 1a, the lag phase ultrasonic treatment can shorten the lag phase of lactobacillus plantarum fermentation in apple juice, and the viable count in the ultrasonic-fermented apple juice is 0.51Log CFU/mL higher than that in the non-ultrasonic-fermented apple juice immediately after 0.5h of ultrasonic treatment. FIG. 1b shows that logarithmic phase ultrasound can accelerate the microbial growth rate, advancing the microbes into stationary phase. Immediately after 0.5h of ultrasonic treatment, the viable count in the ultrasonically fermented apple juice is 0.31Log CFU/mL higher than that in the non-ultrasonically fermented sample. Whereas stationary phase ultrasound had no significant effect on microbial growth as shown in figure 1 c. The influence of the ultrasound in different growth periods on the growth of microorganisms in the apple juice fermentation process is different, on one hand, the physiological states of the microorganisms in different growth periods are different, and the sensitivity to the ultrasound stimulation is different, and on the other hand, the fermentation environments of the microorganisms in different growth periods are greatly different. The ultrasonic treatment can modify the lactobacillus plantarum cell membrane to form repairable pores, thereby enhancing the cell membrane permeability, being beneficial to the transmission of nutrient substances and the adjustment of intracellular pH, and further promoting the growth of microorganisms. The lack of significant effect of ultrasound during the stationary phase may be due to the fact that, after the stationary phase, lactic acid accumulation or nutrient deficiency fundamentally limits the growth of lactobacillus plantarum.
The contents of malic acid, lactic acid and phenolic substances and the in vitro antioxidant activity in the fermentation process of apple juice of example 2 (logarithmic phase ultrasound) and comparative example 1 were tested and the results were as follows:
(1) Malic acid and lactic acid content in ultrasonic-assisted fermentation and non-ultrasonic fermentation apple juice fermentation process
TABLE 1 comparison of malic and lactic acid content (mg/L) during fermentation in example 2 with comparative example 1
Note: capital letters differ significantly for samples at different fermentation times (p < 0.05), and lower case letters differ significantly for samples at different treatments (p < 0.05). In example 2, the logarithmic phase sonication refers to sonication after 8 hours of fermentation.
As can be seen from Table 1, immediately after the logarithmic phase sonication, i.e., at 8.5 hours of fermentation, the malic acid content in the ultrasonically-fermented apple juice was 178.81mg/L lower than that of the non-ultrasonically-fermented apple juice, and the lactic acid content was 233.14mg/L higher than that of the non-ultrasonically-fermented apple juice. The ultrasonic wave can promote the generation of malic acid lactic acid conversion, which is probably from the fact that the ultrasonic wave can promote the growth of microorganisms, enhance the permeability of cell membranes and accelerate the mass transfer inside and outside cells.
(2) Phenolic substance content in ultrasonic-assisted fermentation and non-ultrasonic fermentation apple juice fermentation processes
TABLE 2 comparison of the phenolic content (mg/L) during fermentation in example 2 with that in comparative example 1
Note: capital letters differ significantly for samples at different fermentation times (p < 0.05), and lowercase letters differ significantly for samples at different treatments (p < 0.05). In example 2, the logarithmic phase sonication refers to sonication 8 hours after fermentation.
Chlorogenic acid can be degraded and converted into caffeic acid in lactic acid fermentation, and the caffeic acid can be converted into substances with stronger antioxidant activity or flavor derivatives under the action of phenolic acid decarboxylase and reductase. During the early stage of fermentation, gallic acid derivatives such as tannin and gallate are hydrolyzed to increase the content of gallic acid and catechin, and during the later stage of fermentation, gallic acid can be decarboxylated to generate pyrogalloc acid with stronger antioxidant activity. Procyanidin B2 is a dimer of epicatechin, which is not easily absorbed in the gastrointestinal tract of human body, but is rapidly absorbed in the small intestine after being decomposed into epicatechin by lactic acid bacteria or directly converted into other small molecular substances. The conversion of phenolic substances in the lactic acid fermentation can enhance the antioxidant activity of the fermented apple juice, improve the flavor and improve the bioavailability of the phenolic substances.
As can be seen from table 2, in the fermentation process, the chlorogenic acid content of the ultrasonic fermentation group is significantly lower than that of the non-ultrasonic fermentation group, and the caffeic acid content of the ultrasonic fermentation group is significantly higher than that of the non-ultrasonic fermentation group immediately after the ultrasonic treatment, which indicates that the ultrasonic treatment promotes the conversion of the chlorogenic acid into the caffeic acid; the content of procyanidine B2 in the ultrasonic fermentation group is obviously lower than that in the ultrasonic-free fermentation group just after the ultrasonic treatment, which indicates that the ultrasonic treatment promotes the catabolism of procyanidine B2; the gallic acid content in the ultrasonic fermentation group is obviously lower than that in the non-ultrasonic fermentation group in the fermentation process, which indicates that the decarboxylation conversion of the gallic acid can be enhanced by ultrasonic treatment. The catechin content of the ultrasonic fermentation group is obviously higher than that of the non-ultrasonic fermentation group in the fermentation process, which indicates that the ultrasonic treatment can promote the hydrolysis of the gallic acid ester substances.
The ultrasonic treatment for promoting the derivation of polyphenol in the fermented apple juice is probably because the cell membrane permeability of microorganisms is improved by modifying the cell membrane structure under the cavitation effect of the ultrasonic; the ultrasonic wave improves the mass transfer rate, thereby accelerating the relevant reaction rate of polyphenol derivatization; secondly, the activity of polyphenol derived related enzyme can be improved by ultrasonic wave; finally, ultrasonic stimulation can modulate the expression of polyphenol derivatization-associated enzyme genes. The research of the subject group finds that after lactobacillus plantarum in the fermented apple juice is subjected to ultrasonic treatment in a logarithmic phase (after fermentation for 8 hours), the expression of genes tanL, ubiD and lp _2953 is up-regulated, wherein the tanL is used for coding tannase and can hydrolyze tannin to generate gallic acid, the UbiD plays an important role in the decarboxylation reaction of gallic acid, only under the condition of the expression of the UbiD, the gallic acid can be decarboxylated to generate pyrogalloc acid, and the lp _2953 codes esterase which can be related to the hydrolysis of chlorogenic acid to generate caffeic acid, and the hydrolysis of gallate to generate gallic acid and catechin.
(3) In vitro antioxidant activity in the fermentation process of ultrasonic-assisted fermentation and non-ultrasonic fermentation apple juice
As can be seen from FIG. 2, after the ultrasonic treatment, the ABTS cationic radical scavenging ability of the ultrasonic fermentation group is significantly higher than that of the ultrasonic-free fermentation group. At the end of fermentation, the antioxidant activity of the ultrasonic fermentation group is 16.9% higher than that of the ultrasonic-free fermentation group, which shows that the ultrasonic treatment can enhance the in vitro antioxidant activity of the fermented apple juice. This is probably because sonication enhances polyphenol derivatization in lactic acid fermentation, which generates more potent antioxidant active substances through hydrolysis, decarboxylation, oxidation and reduction reactions, thereby enhancing the antioxidant activity of fermented apple juice.
The lactic acid content, phenolic substance content and in vitro antioxidant activity in the apple juice fermentation processes of example 1 (lag phase ultrasound) and comparative example 1 were tested and the results were as follows:
(1) Malic acid and lactic acid content in ultrasonic-assisted fermentation and non-ultrasonic fermentation apple juice fermentation process
TABLE 3 comparison of malic acid and lactic acid contents (mg/L) during fermentation in example 1 with that in comparative example 1
Note: capital letters differ significantly for samples at different fermentation times (p < 0.05), and lower case letters differ significantly for samples at different treatments (p < 0.05). The delayed phase ultrasound in example 1 refers to ultrasound after 0h of fermentation.
As can be seen from Table 3, in the fermentation process after the delayed ultrasonic treatment, the lactic acid content of the ultrasonic fermentation group is significantly higher than that of the ultrasonic-free fermentation group, and the malic acid content of the ultrasonic fermentation group is significantly lower than that of the ultrasonic-free fermentation group, which indicates that the delayed ultrasonic treatment can also promote the conversion of malic acid into lactic acid. After 6h of ultrasonic treatment, namely fermentation for 6.5h, the content of lactic acid in the 93.6W/L ultrasonic apple juice is 298.2mg/L higher than that of the ultrasonic-free apple juice, and the content of malic acid is 110.36mg/L lower than that of the ultrasonic-free apple juice.
(2) Phenolic substance content in ultrasonic-assisted fermentation and non-ultrasonic fermentation apple juice fermentation processes
TABLE 4 comparison of the phenolic content (mg/L) during fermentation in example 1 with that in comparative example 1
Note: capital letters differ significantly for samples at different fermentation times (p < 0.05), and lower case letters differ significantly for samples at different treatments (p < 0.05). The delayed phase ultrasound in example 1 refers to ultrasound after 0h of fermentation.
As can be seen from table 4, in the fermentation process after the lag phase ultrasonic treatment, the chlorogenic acid content in the ultrasonic fermentation group is significantly lower than that in the ultrasonic-free fermentation group, and the caffeic acid content in the ultrasonic fermentation group is significantly higher than that in the ultrasonic-free fermentation group, which indicates that the lag phase ultrasonic treatment promotes the conversion of chlorogenic acid to caffeic acid; the content of procyanidine B2 in the ultrasonic fermentation group is obviously lower than that in the ultrasonic-free fermentation group just after the ultrasonic treatment, which shows that the ultrasonic treatment in the lag phase promotes the catabolism of procyanidine B2; the gallic acid content in the ultrasonic fermentation group is obviously lower than that in the non-ultrasonic fermentation group in the fermentation process, which indicates that the decarboxylation conversion of the gallic acid can also be enhanced by the ultrasonic treatment in the lag phase. The catechin content of the ultrasonic fermentation group is obviously higher than that of the ultrasonic-free fermentation group in the fermentation process, which indicates that the delayed-phase ultrasonic can promote the hydrolysis of gallic acid ester substances. (3) In vitro antioxidant activity in the process of ultrasonic-assisted fermentation and non-ultrasonic fermentation of apple juice
As can be seen from FIG. 3, the ABTS cationic free radical scavenging ability of the ultrasonic fermentation group is significantly higher than that of the ultrasonic-free fermentation group after the 6 th hour of the delayed-phase ultrasonic treatment, which indicates that the delayed-phase ultrasonic treatment can also enhance the in vitro antioxidant activity of the fermented apple juice.
Comparison of lactic acid content, phenolic content and in vitro antioxidant activity during fermentation of apple juice of comparative example 2 (stationary phase sonication) and comparative example 1, results are as follows:
as is clear from Table 5, table 6 and FIG. 4, the contents of lactic acid, chlorogenic acid, caffeic acid, procyanidin B2, catechin, gallic acid and ABTS. In the stationary phase ultrasonic fermentation group were compared with those in the lag phase and logarithmic phase ultrasonic treatments + The scavenging capacity of free radicals has no obvious difference with that of non-ultrasonic fermentation, namely, the stable-period ultrasonic has little influence on the synthesis of lactic acid, the derivatization of polyphenol such as chlorogenic acid, caffeic acid, procyanidine B2, catechin, gallic acid and the like and the antioxidant activity of apple juice in the process of fermenting apple juice by lactobacillus plantarum.
TABLE 5 comparison of malic acid and lactic acid content (mg/L) during fermentation of comparative example 2 and comparative example 1
Note: capital letters differ significantly for samples at different fermentation times (p < 0.05), and lower case letters differ significantly for samples at different treatments (p < 0.05). Stationary phase ultrasonic fermentation in comparative example 2 refers to fermentation after 18 h.
TABLE 6 comparison of the phenolic content (mg/L) during fermentation in example 1 with that in comparative example 1
Note: capital letters differ significantly for samples at different fermentation times (p < 0.05), and lower case letters differ significantly for samples at different treatments (p < 0.05). Stationary phase ultrasonic fermentation in comparative example 2 refers to fermentation after 18 h.
The embodiments of the present invention have been described above in detail, but this is only an example for the convenience of understanding and should not be construed as limiting the scope of the present invention. Also, various equivalent changes or substitutions are possible for those skilled in the art according to the technical solution of the present invention and the description of the preferred embodiment thereof, but all such changes or substitutions shall fall within the protection scope of the claims of the present invention.
Claims (7)
1. A method for assisting lactobacillus plantarum in fermenting apple juice and simultaneously promoting malic acid-lactic acid conversion and polyphenol derivation by ultrasonic treatment, characterized in that lactobacillus plantarum (A)Lactobacillus plantarum) The method comprises the following steps of:
(1) Preparing a seed culture medium and apple juice;
(2) Inoculating lactobacillus plantarum to a seed culture medium to activate strains;
(3) Inoculating the activated strain into apple juice, and measuring the growth curve;
(4) Inoculating the activated strain into apple juice, performing ultrasonic treatment, and continuously culturing the treated apple juice to obtain a fermented apple juice product;
wherein, the preparation method of the apple juice in the step (1) comprises the following steps: selecting a mature red Fuji apple with excellent properties as a raw material, cleaning and airing, removing peel and fruit core, cutting, crushing apple blocks by using a beater, adding 0.1-0.5% of D-sodium erythorbate and 10% of purified water while crushing, filtering obtained apple pulp by using gauze to remove precipitates, then centrifuging to obtain a supernatant to obtain apple juice, adjusting the pH to 6.0, adding 0.1-0.8% of yeast extract powder, and sterilizing for later use;
the ultrasonic treatment method and conditions in the step (4) are as follows: according to the measurement result of the growth curve, carrying out ultrasonic treatment on the lactobacillus plantarum in the apple juice in a stagnation phase or a logarithmic phase of growth, wherein the ultrasonic power density is 93.6W/L, the ultrasonic frequency is 20kHz, the ultrasonic time is 30min, and the ultrasonic pulse sounding condition is 5s intermittent 5 s;
wherein the stagnation period refers to 0h after inoculation and fermentation; the logarithmic phase means 8h after inoculation and fermentation;
wherein, the steps and conditions for inoculating the activated strains in the step (3) and the step (4) into the apple juice are as follows: inoculating the seed liquid into apple juice at an inoculation amount of 2%, wherein the initial bacterial count is 7.0Log CFU/mL, and fermenting at 30 deg.C.
2. The method for assisting lactobacillus plantarum fermentation apple juice with simultaneous promotion of malic acid-lactic acid conversion and polyphenol derivatization according to claim 1, wherein the main component of the seed culture medium in step (1) is MRS broth prepared by the following steps: commercially available MRS broth 49.3g was weighed, dissolved in 1L water and autoclaved at 121 ℃ for 20min.
3. The method for promoting lactic acid conversion and polyphenol derivatization of malic acid by lactobacillus plantarum fermented apple juice with ultrasonic treatment assistance according to claim 1, wherein the gauze has a mesh size of 120 meshes.
4. The method for promoting both lactic acid conversion malate and polyphenol derivatization in apple juice by lactobacillus plantarum assisted by ultrasonic treatment according to claim 1, wherein the centrifugation conditions are as follows: 4000 Centrifuge at r/min for 20min.
5. The method of claim 1, wherein the sterilization conditions comprise: 85. sterilizing at deg.C for 20min.
6. The method for promoting the lactic acid conversion of malic acid and the derivatization of polyphenol by lactobacillus plantarum fermentation assisted by ultrasonic waves according to claim 1, wherein the growth curve obtained in step (3) is determined by: selecting Lactobacillus plantarum, inoculating to 50mL sterile MRS broth, and culturing at 37 deg.C for 24h; inoculating the cultured bacterial liquid into apple juice according to the inoculation amount of 2%, controlling the temperature to be 30 ℃ by adopting a temperature-controlled bacterial growth tester, and regularly measuring the light absorption value of the apple juice under 600 nm; and drawing a growth curve by taking the light absorption value as the y axis and the culture time as the x axis.
7. The method for promoting the lactic acid conversion of malic acid and the derivatization of polyphenol by lactobacillus plantarum assisted by ultrasonic treatment of apple juice according to claim 1, wherein the apple juice in step (4) is continuously cultured under the following conditions: the sample after ultrasonic treatment is placed in an incubator at 30 ℃ and then is subjected to static culture for 24h.
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