Acid-resistant probiotic compound fermentation method and application of acid-resistant probiotic compound fermentation method in blueberry juice

Abstract

The invention discloses a compound fermentation method of acid-resistant probiotics and application of the acid-resistant probiotics in blueberry juice, and particularly relates to compound fermentation of plant lactobacillus B4 and plant lactobacillus SS6 and application of the compound fermentation method. The two strains are preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation numbers of the strains are respectively CGMCC NO.30881 and CGMCC NO.30880. The invention also discloses a preparation method of the strain. The viable count of the lactobacillus plantarum still exceeds 6 log CFU / ml after the lactobacillus plantarum is fermented for 72 hours in blueberry juice (pH = 3.0), and is two orders of magnitude higher than that of a common commercial strain (lactobacillus plantarum LP39); and the two strains are subjected to compound fermentation, so that the total phenol content, DPPH free radical scavenging rate and terpenoid content of the blueberry juice can be increased at the same time, and the influence of fermentation on the color of the blueberry juice can be reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of fruit juice and microorganisms, and in particular to a method for compound fermentation of acid-resistant probiotics and application thereof in fermenting blueberry juice. Background Art

[0002] Blueberries, a small berry belonging to the genus Vaccinium in the Ericaceae family, are rich in nutritional value, packed with antioxidants such as flavonols, anthocyanins, and phenolic acids. They also boast benefits such as protecting vision and boosting immunity, earning them the reputation of a "superfruit." However, blueberries primarily ripen and are harvested in the summer, making them susceptible to mechanical damage and microbial degradation. Therefore, processed blueberry products have become an effective means of extending the supply chain and increasing added value. Currently, the main blueberry products on the market include blueberry juice, blueberry wine, and dried blueberries.

[0003] In recent years, lactic acid bacteria have been widely used in fruit juices due to their excellent fermentation properties, such as improving the nutritional quality, flavor properties, antioxidant activity, antibacterial activity and shelf life of food, as well as reducing adverse compounds. Patent 202110435091.1 discloses two strains of Lactobacillus and their use in fermented blueberry juice. The filtered blueberry juice is adjusted to pH 4.0, and its soluble solids content is adjusted to 13°Brix with glucose. Then, lactic acid bacteria are inoculated and fermented at 37°C for 48 hours to obtain fermented blueberry juice. However, the lactic acid bacteria used in this patent are strains of different species, and pH and Brix adjustment are required, which adds a pre-treatment step and is not conducive to industrial and efficient production. Therefore, it is necessary to develop a more convenient and efficient technology.

[0004] Lactobacillus plantarum's excellent antioxidant and antibacterial properties, tolerance to acidic pH, gastrointestinal resistance, and adhesion to the intestinal mucosa enable this LAB to have beneficial effects on host health. The probiotic functions of different L. plantarum strains are diverse, and their growth and metabolism in juice vary significantly, resulting in juice products exhibiting different nutritional and flavor characteristics. Furthermore, mixed inoculation of strains may further affect the quality characteristics of juice. Currently, there is limited research on the effects of different L. plantarum strains and their inoculation methods on blueberry juice quality, and the variety of related products is relatively limited.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The present invention aims to provide a method for fermenting blueberry juice using a composite acid-resistant probiotic fermentation system and its application in fermenting blueberry juice. The strains used in this method have excellent acid resistance (pH 3.0-3.2), can increase the total phenol content, DPPH free radical scavenging rate, and terpenoid content in blueberry juice, and can reduce the impact of fermentation on color.

[0007] A Lactobacillus plantarum B4 is deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 7, 2024, and the deposit number is CGMCC NO.30881.

[0008] A Lactobacillus plantarum strain SS6 was deposited in the General Microbiology Center of the China Culture Collection Administration, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date was June 7, 2024, and the deposit number was CGMCC NO.30880.

[0009] The present invention provides a probiotic compound fermentation method and application thereof in improving the quality of blueberry juice, characterized in that the method comprises Lactobacillus plantarum B4 and Lactobacillus plantarum SS6.

[0010] The present invention provides an application for increasing the number of viable bacteria in low pH (3.0-3.2) fermented juice, characterized in that the method comprises Lactobacillus plantarum B4 and / or Lactobacillus plantarum SS6.

[0011] In the present invention, the quality improvement includes increasing the total phenols, DPPH free radical scavenging rate, terpenoid content and reducing the impact on juice color.

[0012] In the embodiment of the present invention, Lactobacillus plantarum B4, SS6 and LP39 were selected for lactic acid fermentation.

[0013] In the embodiment of the present invention, the single bacteria inoculation is specifically as follows: the activated Lactobacillus plantarum is inoculated with 10 6-8 CFU / mL inoculated into blueberry juice and fermented at 28-35°C for 72h; the mixed inoculation is specifically as follows: the two activated plant lactobacillus strains are inoculated at a ratio of 1:1, with a total concentration of 10 6-8 CFU / mL was inoculated into blueberry juice and fermented at 28-35℃ for 72h. Preferably, the inoculation concentration was 3×10 7 CFU / mL, and the fermentation temperature was 30℃.

[0014] In the embodiment of the present invention, the blueberry variety of the blueberry juice is "Lanfeng".

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] (1) The present invention can increase the viable bacterial count in low pH (3.0-3.2) fermented juice. After 72 hours of fermentation of Lactobacillus plantarum B4 and Lactobacillus plantarum SS6 in blueberry juice with an initial pH of 3.0, the viable bacterial count of the blueberry juice was 6.73 log CFU / mL.

[0017] (2) The present invention can significantly increase the total phenol content, DPPH free radical scavenging rate and terpenoid content in blueberry juice, and reduce the effect of fermentation on the color of blueberry juice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The changes of viable bacteria count of single strains in fermented blueberry juice of different treatment groups;

[0019] Figure 2 Table of color characteristic parameters of fermented blueberry juice in different treatment groups;

[0020] Figure 3 The total phenolic content in fermented blueberry juice of different treatment groups;

[0021] Figure 4 is the DPPH free radical scavenging rate in fermented blueberry juice of different treatment groups;

[0022] Figure 5 is the peak area ratio of terpenoid compounds in fermented blueberry juice of different treatment groups. DETAILED DESCRIPTION

[0023] Example 1 Blueberry juice fermentation

[0024] The present invention involves three strains of Lactobacillus plantarum: B4, SS6, and LP39. B4 and SS6 are deposited at the General Microbiology Center of the China National Center for Microbiological Culture Collection, with deposit numbers CGMCC NO. 30881 and CGMCC NO. 30880, respectively; LP39 is a commercial strain purchased from the China National Center for Industrial Microbiological Culture Collection.

[0025] The treatment groups set up in the present invention are as follows: initial group (original juice), blank group (CK), B4 group, LP39 group, SS6 group, B4+LP39 group, B4+SS6 group, LP39+SS6 group.

[0026] The initial group (original juice) is blueberry juice that has just been sterilized; the blank group (CK) is a blank control group that is not inoculated with Lactobacillus plantarum; the B4 group is blueberry juice inoculated with Lactobacillus plantarum B4 alone; the LP39 group is blueberry juice inoculated with Lactobacillus plantarum LP39 alone; the SS6 group is blueberry juice inoculated with Lactobacillus plantarum SS6 alone; the B4+LP39 group is blueberry juice inoculated with a mixture of Lactobacillus plantarum B4 and LP39; the B4+SS6 group is blueberry juice inoculated with a mixture of Lactobacillus plantarum B4 and SS6; and the LP39+SS6 group is blueberry juice inoculated with a mixture of Lactobacillus plantarum LP39 and SS6.

[0027] Experimental method: After the frozen blueberries were thawed at room temperature, they were crushed and sulfurized, pectinase was added, and after cold maceration for 24 hours, the juice was squeezed out, filtered through a filter, and sterilized at high temperature before being dispensed into 100ml conical bottles for use. The activated Lactobacillus plantarum B4, SS6, and LP39 were inoculated with 3×10 7 Blueberry juice was inoculated with a concentration of 100 CFU / mL and fermented at 30°C for 72 hours to prepare fermented blueberry juice samples. Viable bacterial counts were determined at 12, 24, 48, and 72 hours of fermentation. The CIE Lab value, DPPH radical scavenging rate, total phenolic compounds, and aroma compounds were also determined for the post-fermentation blueberry juice.

[0028] Example 2 Changes in viable bacteria count in each treatment group

[0029] Real-time quantification of viable bacteria using PMA-CELL-qPCR was used to monitor changes in viable bacterial counts in each treatment group during fermentation. The reaction system is shown in Table 1. The qPCR protocol (SYBR®) was as follows: pre-denaturation at 95°C for 15 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds, followed by annealing / extension at 60°C for 20 seconds. The primers used are shown in Table 2.

[0030] Table 1 PMA-CELL-qPCR reaction system

[0031]

[0032] Table 2 qPCR primers for three bacterial strains

[0033]

[0034]

[0035] The changes in the number of viable bacteria of each strain in all treatment groups are shown in Figure 1In the single inoculation treatment group, the viable cell count of B4 group ranged from 5.83±0.45 to 7.45±0.05 lg CFU / mL; the viable cell count of SS6 group ranged from 5.93±0.04 to 7.15±0.04 lg CFU / mL; the viable cell count of LP39 group ranged from 4.03±0.02 to 6.04±0.44 lg CFU / mL. At the end of fermentation, the viable cell count of LP39 group was 4.03±0.02 lg CFU / mL; the viable cell count of B4 group was 6.48±0.00 lg CFU / mL; the viable cell count of SS6 group was 6.27±0.27 lg CFU / mL. In the mixed inoculation treatment group, the viable cell count of B4+LP39 group ranged from 5.83±0.06 to 6.66±0.14 lg CFU / mL; the viable cell count of LP39+SS6 group ranged from 5.00±0.12 to 6.38±0.52 lg CFU / mL; the viable cell count of B4+SS6 group ranged from 5.35±0.03 to 7.47±0.33 lg CFU / mL. At the end of fermentation, the viable cell count of B4+LP39 group was 6.29±0.05 lg CFU / mL; the viable cell count of LP39+SS6 group was 5.51±0.15 lg CFU / mL; the viable cell count of B4+SS6 group was 6.73±0.18 lg CFU / mL. Figure 1 It was shown that, in both single inoculation and mixed inoculation groups, the viable cell count of commercial strain LP39 was significantly lower than that of B4 and SS6, and the viable cell count of B4 and SS6 mixed inoculation group was the highest at the end of fermentation, which was 2 orders of magnitude higher than that of single inoculation of commercial strain LP39, and met the requirement of national standard that the viable cell count of live lactic acid bacteria beverage should be greater than 10 6 CFU / mL. Therefore, mixed inoculation of B4 and SS6 could significantly improve the viable cell count in low pH (3.0-3.2) fermented juice.

[0036] Example 3 Determination of color, total phenol content, DPPH free radical scavenging rate and terpenoid compounds of fermented blueberry juice

[0037] 1. CIE Lab determination of blueberry juice

[0038] The determination of blueberry juice color referred to the method used by Wang et al. After filtering the sample to be tested, the absorbance A440, A530, A600 at 440 nm, 530 nm and 600 nm was measured using a spectrophotometer. The lightness (L*), red-green hue (a*), yellow-blue hue (b*), chroma (C*), chroma angle (h*), color difference (△E*) were calculated by formula. The calculation results were shown in Table 3. Figure 2 .

[0039] b* represents yellow-blue hue, b* value generally increased after inoculation, indicating that the yellow hue of blueberry juice is enhanced, and the blue hue is weakened. Blue hue is one of the characteristics of blueberry, which is particularly important for the overall color of the juice. Compared with the non-inoculated blueberry juice (blank group) and different treatment groups: except for the LP39 group, the rest of the groups will reduce the blue hue of blueberry juice further loss, and the b* of SS6 group is the smallest, indicating that SS6 has the strongest protective effect on the blue hue of blueberry juice. ΔE is defined as the total color difference of the sample, the larger the ΔE value, the greater the color difference. Compared with the non-inoculated blueberry juice (blank group) and different treatment groups: SS6 group and B4+SS6 group have the smallest effect on the color difference of blueberry juice.

[0040] 2. Total phenol determination

[0041] Reference The total phenol content in blueberry juice was determined by the method of Zhang et al., and the determination results are shown in Table 2. Figure 3 The total phenol content of the initial group (original juice) was 486.42±1.01 mg / L; the total phenol content of the blank group (CK) was 471.68±2.73 mg / L; at the end of fermentation, the total phenol content of the LP39 group was 483.53±0.61 mg / L; the total phenol content of the B4 group was 474.58±2.43 mg / L; the total phenol content of the SS6 group was 474.84±5.17 mg / L; the total phenol content of the B4+LP39 group was 480.63±2.13 mg / L; the total phenol content of the LP39+SS6 group was 480.50±2.29 mg / L; the total phenol content of the SS6+B4 group was 502.21±21.57 mg / L. Compared with the non-inoculated blueberry juice (blank group) and different treatment groups: SS6, B4 mixed inoculation can significantly improve the total phenol content of blueberry juice, and is 18.68 mg / L higher than that of single inoculation of commercial strain LP39.

[0042] 3. DPPH free radical scavenging rate determination

[0043] The DPPH free radical scavenging rate of blueberry juice in different treatment groups was determined by using the DPPH free radical scavenging ability detection kit of Boxbio, and the results are shown in Table 3. Figure 4The DPPH radical scavenging rates of the initial group (original juice) were 92.13±0.88%; those of the blank group (CK) were 89.38±0.04%. At the end of fermentation, the DPPH radical scavenging rates of the LP39 group, the B4 group, the SS6 group, the B4+LP39 group, the LP39+SS6 group, and the SS6+B4 group were 94.94±0.95%, 93.70±2.15%, and 96.19±0.86%, respectively. The results indicate that inoculation with Lactobacillus plantarum can enhance the DPPH radical scavenging rate of blueberry juice.

[0044] 4. Determination of Terpenoids

[0045] Terpenoids in blueberry juice were determined using HS-SPME-GC-MS. GC-MS conditions included an HP-INNOwax 60 m × 0.25 mm × 0.25 μm column (J&W Scientific, USA). The carrier gas was high-purity helium at a flow rate of 1 mL / min. The inlet temperature was 250°C, splitless injection mode was used, and the desorption time was 8 min. The temperature program was 50°C for 1 min, then increased to 220°C at a rate of 3°C / min and held for 5 min. The mass spectrometer ionization mode was EL, with an ion source temperature of 230°C, an ionization energy of 70 eV, a quadrupole temperature of 150°C, an interface temperature of 280°C, and a mass scan range of 30–350 μm. Compound identification was performed using Automated Mass Spectral Deconvolution and Identification Software (AMDIS). The compound ion fragment information was extracted by MSD ChemStation (Agilent Technologies, USA) and the peak area of ​​each compound was determined. The obtained compound peak area was divided by the peak area of ​​the internal standard compound (4-methyl-2-pentanol, ion fragment 45) for semi-quantification. The determination results are shown in Figure 5 .

[0046] Terpenes contribute to aroma properties such as sweet, floral, and fruity, and are the primary aroma compounds in blueberry juice. Studies have reported that inoculation with lactic acid bacteria can increase terpenoid content and positively impact the juice's sensory properties. A total of 33 terpenoids were detected in all fermented blueberry juices, 23 of which showed significant differences between samples. Comparison of the uninoculated blueberry juice blank (CK) with the different treatments revealed that all treatments inoculated with Lactobacillus plantarum significantly increased terpenoid content in the blueberry juice. Compared with the blank group, the B4 group can significantly increase the content of 23 terpenoid compounds, with a change fold range of 2.23 to 19.20; the SS6 group can significantly increase the content of 16 terpenoid compounds, with a change fold range of 1.50 to 11.13; the LP39 group can significantly increase the content of 16 terpenoid compounds, with a change fold range of 1.12 to 14.25; the B4+LP39 group can significantly increase the content of 21 terpenoid compounds, with a change fold range of 1.65 to 16.87; the LP39+SS6 group can significantly increase the content of 14 terpenoid compounds, with a change fold range of 1.40 to 11.06; the B4+SS6 group can significantly increase the content of 17 terpenoid compounds, with a change fold range of 1.66 to 14.05, and can additionally increase the content of 1,3,8-p-Menthatriene compared with the commercial strain LP39 alone.

[0047] The above results show that mixed inoculation of B4 and SS6 has the least effect on the color of blueberry juice, and can significantly increase the total phenol content, DPPH free radical scavenging rate and terpenoid content of blueberry juice. Its comprehensive performance in improving the quality of blueberry juice is better than that of the commercial strain LP39.

[0048] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention.

Claims

1. A fruit-derived fermentation strain of Lactobacillus plantarum B4, deposited in the China General Microbiology Center (CGMCC) under the China Culture Collection Administration, with a deposit number of CGMCC No. 30881. The depository address is the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

2. A fruit-derived fermentation strain of Lactobacillus plantarum SS6, deposited in the China General Microbiology Center (CGMCC) under the China Culture Collection Administration, with the deposit number CGMCC No. 30880. The depository address is the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.

3. A probiotic compound fermentation method, characterized in that: The method comprises the Lactobacillus plantarum B4 of claim 1 and the Lactobacillus plantarum SS6 of claim 2.

4. Use of the plant lactobacillus as described in claims 1-3 in increasing the viable count of low pH (3.0-3.2) fermented juice.

5. The use according to claim 4, wherein the fruit juice is blueberry juice (pH=3.0).

6. Use of the plant lactobacillus according to claims 1-3 in increasing the total phenol content, DPPH free radical scavenging rate, terpenoid content and reducing color impact of fermented juice.

7. The use according to claim 6, characterized in that The inoculation is as follows: the activated Lactobacillus plantarum is inoculated with 10 6-8 CFU / mL was inoculated into blueberry juice and fermented at 28-35°C for 72h. Preferably, Lactobacillus plantarum B4 and SS6 were mixed and inoculated in a ratio of 1:1, and the inoculation concentration of Lactobacillus plantarum was 3×10 7 CFU / mL, and the fermentation temperature was 30℃.

8. The juice as claimed in claims 6-7 is blueberry juice (pH=3.0).

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