New application of bacillus lactis DU-106 in color protection of anthocyanin and color protection method of anthocyanin-rich fruit juice

Abstract

The invention belongs to the field of food preparation, and particularly relates to a novel application of bacillus lactis DU-106 in color protection of anthocyanin and a color protection method of anthocyanin-rich fruit juice. The invention provides a novel application of bacillus lactis DU-106 in color protection of anthocyanin. The bacillus lactis DU-106 is preserved in the Guangdong Microbial Culture Collection Center on March 29, 2019, and the preservation number is GDMCC NO.1. 1581. The invention also provides a preparation method of the bacillus lactis DU-106. The bacillus lactis DU-106 has the advantages that the bacillus lactis DU-106 is preserved in the Guangdong Microbial Culture Collection Center on March 29, 2019; a color protection system constructed through fermentation of the bacillus lactis DU-106 can effectively maintain the structural stability of anthocyanin, and color protection of anthocyanin-rich food is realized. Meanwhile, the method has edible safety, breaks through the limitation of traditional chemical color protection, and is of great significance to food development.

Description

Technical Field

[0001] The invention belongs to the field of food preparation, and particularly relates to a new application of lactic acid bacillus DU-106 in anthocyanin color protection and a color protection method for anthocyanin-rich juice. Background Art

[0002] Anthocyanins, also known as flower pigments, are a class of water-soluble pigments found widely in plants in nature and belong to the flavonoid class of compounds. Anthocyanins are powerful antioxidants with multiple physiological functions, including scavenging free radicals in the body, slowing aging, and preventing chronic diseases such as cancer and cardiovascular disease. They can also improve vision, protect the cardiovascular system, enhance immunity, and improve skin health. However, anthocyanins are relatively unstable. Natural or processed foods rich in anthocyanins are easily affected by factors such as pH, temperature, oxidation, and metal ions during processing and storage, resulting in a decrease in color and nutritional value. Traditional color protection methods often use vitamin C or citric acid as color preservatives, but these color preservatives are inherently unstable and easily affected by factors such as light and high temperature, affecting product safety and consumer acceptance. Summary of the Invention

[0003] To solve the above technical problems, the present invention provides a new application of Lactobacillus DU-106 in anthocyanin color protection and a color protection method for anthocyanin-rich fruit juice. The present invention utilizes Lactobacillus DU-106 to ferment and protect anthocyanins. Its bacteria and fermentation products can effectively maintain the structural stability of anthocyanins. Lactic acid and other substances can be continuously produced during the fermentation process, which can achieve a long-term color protection effect on anthocyanins. Since Lactobacillus DU-106 is a recognized probiotic, the method provided by the present invention can not only effectively protect the color, but also has food safety, providing a new color protection solution for the preparation of anthocyanin-rich foods.

[0004] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a lactic acid bacillus ( Bacillus lactics ) A new application of DU-106 in anthocyanin color protection. The lactic acid Bacillus DU-106 was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 29, 2019, with the deposit number GDMCC NO.1.1581.

[0005] Lactobacillus ( Bacillus lacticsDU-106 is a probiotic isolated from fermented yogurt in Inner Mongolia. This strain was previously published in "Identification and Comparative Genomic Analysis of a Highly Lactic Acid-Producing Lactic Acid Bacillus DU-106" (Master's thesis, Xueyin Wu). This strain exhibits excellent lactic acid synthesis during fermentation. Through efficient acid production, it creates an acidic environment to stabilize anthocyanin structures. It also secretes antioxidants (such as polysaccharides and glutathione) to inhibit oxidative degradation, significantly improving the stability of anthocyanins in foods. This approach breaks through the limitations of traditional chemical color protection and provides a highly effective and safe biological solution for the stabilization of natural pigments and the development of functional foods.

[0006] Preferably, the novel application is in the preparation of anthocyanin-enriched beverages.

[0007] The anthocyanin-rich beverage described in the present invention refers to an anthocyanin-rich beverage obtained by exogenously adding anthocyanins or a beverage made by squeezing / extracting natural fruits or vegetables containing high concentrations of anthocyanins, including but not limited to blueberry juice, black currant juice, wolfberry juice and purple carrot juice.

[0008] Lactic acid Bacillus DU-106 possesses the unique heat and acid resistance of the Bacillus genus, demonstrating its strong adaptability and stability during food processing. It effectively maintains the stability of anthocyanins in anthocyanin-rich juices, maintaining their vibrant color during preparation and long-term storage. This overcomes the limitations of traditional color preservation technologies that rely on chemical additives, while also significantly improving the juice's taste.

[0009] In a second aspect, the present invention further provides a method for protecting the color of anthocyanin-rich juice, the preparation method comprising the following steps: Step 1: preparing anthocyanin-rich juice, and sterilizing the anthocyanin-rich juice; Step 2: Inoculate the above-mentioned Lactobacillus DU-106 into the anthocyanin-rich juice after sterilization in step 1, with the inoculation amount ≥ 1×10 5 CFU / mL, fermentation was terminated at a temperature of 30-45°C and pH ≤ 4.7.

[0010] In step 1, the anthocyanin-rich juice is sterilized. The sterilization method selected includes, but is not limited to, pasteurization, combined temperature-pressure sterilization, or other non-thermal sterilization methods. The sterilization method that has the least impact on the anthocyanin content and juice quality can be selected based on actual results.

[0011] In step 2, the juice is inoculated with the aforementioned Lactobacillus DU-106 and then fermented, producing a large amount of acid over 24-48 hours, creating an acidic environment and protecting the color of the juice. The Lactobacillus DU-106 strain employed in the present invention has an optimal growth and acid production temperature of 30-45°C.

[0012] Preferably, the sterilization treatment in step 1 is a low-temperature and long-term sterilization method.

[0013] Low-temperature, long-term sterilization (LTLT), as a traditional thermal sterilization method, is mainly used for the sterilization of liquid foods such as milk and juice. The generally used sterilization temperature is 62-65°C and the time is 20-30 minutes.

[0014] More preferably, the sterilization temperature is 63° C. and the time is 30 min.

[0015] Preferably, the anthocyanin-rich juice is wolfberry juice, and its preparation method is: adding water to dried wolfberries for rehydration and then squeezing the juice, and separating the solid and liquid to obtain wolfberry juice; or adding water to fresh wolfberries for squeezing the juice and then separating the solid and liquid to obtain wolfberry juice; or squeezing the fresh wolfberries for solid and liquid separation to obtain wolfberry juice; or extracting dried wolfberries / fresh wolfberries with water and then separating the solid and liquid to obtain wolfberry juice.

[0016] More preferably, the wolfberry dried fruit is black wolfberry dried fruit; and the mass volume ratio of the black wolfberry dried fruit to water is 1:20 (g / mL).

[0017] Black wolfberry is rich in anthocyanins, proanthocyanidins, polysaccharides and trace elements, and is one of the berries with the highest anthocyanin content.

[0018] Preferably, the access volume is ≥1×10 6 CFU / mL.

[0019] Preferably, the temperature is 37°C.

[0020] In a third aspect, the present invention also provides anthocyanin-rich juice prepared according to the above color protection method.

[0021] Preferably, the anthocyanin-rich juice is anthocyanin-rich black wolfberry juice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 The pH value of the black wolfberry juice in Experimental Example 1 of the present invention at different fermentation days; Figure 2 The anthocyanin preservation rate of black wolfberry juice in Experimental Example 1 of the present invention at different fermentation days; Figure 3The color value change (a*) of the black wolfberry juice in Experimental Example 1 of the present invention at different fermentation days; Figure 4 The color value change (b*) of the black wolfberry juice in Experimental Example 1 of the present invention at different fermentation days; Figure 5 The color value changes (L*) of the black wolfberry juice in Experimental Example 1 of the present invention at different fermentation days; Figure 6 The color value change (ΔE) of the black wolfberry juice in Experimental Example 1 of the present invention at different fermentation days; Figure 7 This is the standard curve of anthocyanins in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0024] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0025] Black wolfberry was purchased from Tianfangjian Co., Ltd.

[0026] The strains used in the examples of the present invention are derived from: Lactobacillus ( Bacillus lactics DU-106: Deposited with the Guangdong Provincial Center for Microbial Culture Collection on March 29, 2019, with the accession number GDMCC No. 1.1581. This strain has been published in "Identification and Comparative Genomic Analysis of a High-Lactic Acid-Producing Lactobacillus DU-106" (Master's thesis, Xueyin Wu).

[0027] Lactobacillus powder is a kind of Lactobacillus ( Bacillus lactics ) DU-106 enrichment culture was followed by freeze-drying to obtain freeze-dried bacterial powder. The number of viable cells of Lactobacillus DU-106 in the bacterial powder was 6×10 7 -8×10 7 CFU / g.

[0028] Example 1 Step 1: Mix the dried wolfberry fruit with water at a mass-to-volume ratio (g / mL) of 1:20. After soaking for 10 minutes and rehydrating, use a juicer to extract the juice. After filtering, take the filtrate to obtain the black wolfberry juice.

[0029] Step 2: Heat the black wolfberry juice obtained in step 1 to 65° C. in a water bath, maintain for 30 minutes, and then quickly cool to below 40° C. to obtain sterilized black wolfberry juice.

[0030] Step 3: The sterilized black wolfberry juice was divided into D1, D2, D3, and D4 groups, and 0.005 g, 0.01 g, 0.015 g, and 0.02 g of Lactobacillus powder were added thereto, respectively. The mixture was placed in a 37°C incubator for fermentation for 5 days, and the fermentation liquid of the black wolfberry juice was collected for measurement every 24 hours.

[0031] 1. pH determination The pH of the fermented broth of black wolfberry juice in groups D1, D2, D3, and D4 was measured at 0, 24, 48, 36, 72, 96, and 120 h of fermentation. Figure 1 shown.

[0032] The results showed that the pH values ​​of all groups decreased over time, indicating that Lactobacillus DU-106 produced lactic acid during fermentation, leading to a decrease in pH. During the first 48 hours of fermentation, the pH value dropped rapidly, and the greater the amount of bacterial powder added, the faster the pH drop. In group D4 (0.02 g of bacterial powder), the pH of the fermentation broth reached 4.6 after 24 hours. After 5 days of fermentation, the pH value of all groups remained below 4.6.

[0033] 2. Determination of anthocyanin preservation rate The absorbance values ​​of the fermented broth of black wolfberry juice in groups D1-D4 at different fermentation times were measured at a wavelength of 510 nm, and the preservation rate was calculated using the following formula: X(%)=A / A0×100; Where: X is the anthocyanin preservation rate, %; A0 is the absorbance of black wolfberry juice at fermentation time 0; A is the absorbance of black wolfberry juice after fermentation.

[0034] Depend on Figure 2 The results show that the anthocyanin content of each group decreased significantly between days 0 and 1 of fermentation. However, as the fermentation time increased, the anthocyanin preservation rate of each group of black wolfberry juice tended to stabilize, indicating that the acidic environment provided by Lactobacillus DU-106 enhanced the stability of anthocyanins in black wolfberry juice. The anthocyanin preservation rates of groups D2, D3, and D4 were relatively stable between days 2 and 5. Combined with pH testing, it was found that the preservation effect of anthocyanins was positively correlated with acidity. Anthocyanins maintained good stability below pH 4.7, and increasing the inoculum size helped achieve this condition more quickly, thus facilitating anthocyanin preservation.

[0035] 3. Color value determination The color values ​​of the fermented black wolfberry juice of groups D1-D4 at different fermentation times, a*, b*, L*, and ΔE, were measured using a colorimeter. Figure 3-6. a* reflects the red and green hue, +a* represents red, and the larger the +a* value, the higher the saturation of the red hue, that is, the purer the red; -a* represents green, and the larger the -a* value, the higher the saturation of the green hue, that is, the purer the green; b* represents the yellow-blue hue, +b* represents yellow, and -b* represents blue. L* represents the lightness value of the color, which refers to the brightness of the color. L*=0 indicates black, L*=100 indicates white, and the closer to 100, the brighter it is. ΔE represents the total color difference, which is calculated from the three dimensions of L*, a*, and b*, and is used to quantify the color difference between the sample and the standard. The smaller the ΔE value, the less obvious the color difference; the larger the ΔE value, the more obvious the color difference.

[0036] As shown in the figure, the a* values ​​of each group increased over time, indicating that certain substances in the black goji berry juice underwent browning during storage. Furthermore, as the concentration of Lactobacillus DU-106 increased, the a* values ​​decreased, with group D4 having the lowest a* value. This indicates that Lactobacillus DU-106 slowed the browning of substances in the fermented black goji berry juice, thereby protecting its color. When ΔE ≈ 2, a color difference observable to the naked eye has occurred. The color difference values ​​for all groups were less than 2, indicating that the color difference was not significant. This indicates that Lactobacillus DU-106 enhances the color stability of black goji berry juice.

[0037] Example 2 This embodiment provides a ruthenic wolfberry juice rich in anthocyanins, and the preparation method thereof comprises the following steps: Step 1: Mix the dried wolfberry fruit with water at a mass-to-volume ratio (g / mL) of 1:20. After soaking for 10 minutes and rehydrating, use a juicer to extract the juice. After filtering, take the filtrate to obtain the black wolfberry juice.

[0038] Step 2: Heat the black wolfberry juice obtained in step 1 to 65° C. in a water bath, maintain for 30 min, and then quickly cool to 40° C. to obtain sterilized black wolfberry juice.

[0039] Step 3: Adding the lactic acid Bacillus powder to the sterilized black wolfberry juice for activation and culture until the stable phase, thereby obtaining activated lactic acid Bacillus DU-106.

[0040] Step 4: Inoculate the activated Lactobacillus DU-106 into the sterilized black wolfberry juice with an inoculum size of 1×10 6 CFU / mL, and then cultured in a 37℃ constant temperature box for 24 h to obtain black wolfberry juice rich in anthocyanins.

[0041] The anthocyanin-rich black wolfberry juice was placed in an aluminum foil bottle for 30 days. The anthocyanin content was measured every 10 days to observe the changes in the content.

[0042] LC-MS was used for detection. Chromatographic conditions included a Thermo ODS HYPERSIL C18 column (250 mm × 4.6 mm), mobile phase A: 3% formic acid in water, mobile phase B: 1% formic acid in acetonitrile, detection wavelength of 530 nm, column temperature of 35°C, injection volume of 25 μL, and gradient elution conditions as shown in Table 1. Mass spectrometry conditions included a mass spectrometry ion scan range of 200–1000, an ESI ion source, detection mode of positive ionization, a nebulizer of 1.5 L / min, a drying gas of 115.0 kPa, and an ion source temperature of 200°C. A standard curve was prepared using petunidin-3-o-glucoside as the standard. See Table 1. Figure 7 The prepared anthocyanin-rich black wolfberry juice was filtered through a 0.22 μm water filter membrane, and the anthocyanin peak area was determined according to the above method. The anthocyanin content was calculated using the standard curve. The results are shown in Table 2.

[0043] Table 1 Gradient elution conditions

[0044] Table 2 Anthocyanin content

[0045] The results show that the anthocyanin content in the anthocyanin-rich black wolfberry juice prepared within 30 days did not change much, maintaining at around 20 ng / mL, indicating that the color protection method provided by the present invention can effectively slow down the decomposition of anthocyanins in black wolfberry juice, prolong its half-life, and thus improve its storage stability.

[0046] In summary, the present invention utilizes Lactobacillus DU-106 to protect anthocyanins, preventing their decomposition during the production process and achieving long-term color preservation through continuous fermentation. Furthermore, Lactobacillus DU-106, as a food-grade probiotic, has excellent safety, making the method provided by the present invention potentially applicable in the food preparation field.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A lactic acid bacillus ( Bacillus lactics ) A new application of DU-106 in anthocyanin color protection, characterized in that: The lactic acid Bacillus DU-106 was deposited in the Guangdong Provincial Microbial Culture Collection Center on March 29, 2019, with the deposit number GDMCC NO.1.1581.

2. The new application according to claim 1, characterized in that New application in the preparation of anthocyanin-rich beverages.

3. A method for protecting the color of anthocyanin-rich juice, characterized in that: The preparation method comprises the following steps: Step 1: preparing anthocyanin-rich juice, and sterilizing the anthocyanin-rich juice; Step 2: Inoculate the Lactobacillus DU-106 of claim 1 into the anthocyanin-rich juice sterilized in step 1, with the inoculation amount being ≥ 1×10 5 CFU / mL, fermentation was terminated at a temperature of 30-45°C and pH ≤ 4.

7.

4. The method for protecting the color of anthocyanin-rich juice according to claim 3, characterized in that: The sterilization method in step 1 is a low-temperature, long-term sterilization method.

5. The method for protecting the color of anthocyanin-rich juice according to claim 3, characterized in that: The anthocyanin-rich fruit juice is wolfberry juice, and its preparation method is as follows: adding water to dried wolfberries for rehydration and then squeezing the juice, and then separating the solid and liquid to obtain wolfberry juice; or adding water to fresh wolfberries for squeezing the juice and then separating the solid and liquid to obtain wolfberry juice; or squeezing the fresh wolfberries for solid and liquid separation to obtain wolfberry juice; or extracting dried wolfberries / fresh wolfberries with water and then separating the solid and liquid to obtain wolfberry juice.

6. The method for protecting the color of anthocyanin-rich juice according to claim 5, characterized in that: The dried wolfberry fruit is black wolfberry dried fruit; the mass volume ratio of the black wolfberry dried fruit to water is 1:20 (g / mL).

7. The method for protecting the color of anthocyanin-rich juice according to claim 3, characterized in that: The access volume is ≥1×10 6 CFU / mL.

8. The method for protecting the color of anthocyanin-rich juice according to claim 3, characterized in that: The temperature was 37°C.

9. Anthocyanin-rich juice prepared according to the color protection method of anthocyanin-rich juice according to claims 3-8.

10. The anthocyanin-rich juice according to claim 9, characterized in that The anthocyanin-rich juice is anthocyanin-rich black wolfberry juice.

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