Method for preparing probiotic blackened jujube juice based on solid-liquid step-by-step green precise fermentation

Abstract

The invention belongs to the technical field of food biology, and relates to blackened jujube juice, in particular to a method for preparing probiotic blackened jujube juice based on solid-liquid step-by-step green precise fermentation. According to the preparation method disclosed by the invention, the probiotic compound combination capable of effectively increasing the viable count is screened and excavated, and the viable count of the probiotics in the product is increased on the basis of retaining rich nutritional ingredients of the blackened jujubes by fermenting the blackened jujube juice, so that the functionality of the product is improved. Through precise fermentation of probiotics, the content of sugar in the blackened jujube juice is further reduced, the content of nutritional ingredients such as organic acid is increased, sugar in the jujube juice is promoted to be efficiently converted into lactic acid (the sugar degradation rate is larger than or equal to 25%) through symbiotic metabolism regulation and control of the two bacteria, meanwhile, key aroma substances such as ethyl acetate and n-hexyl alcohol are generated, and the product is endowed with fresh fruity flavor and low-sugar characteristics; therefore, the flavor and taste of the beverage are improved, and the blackened jujube juice has higher biological functionality; and through the hypoglycemic effect of the probiotics, a healthier beverage choice is provided for people with low sugar requirements, and the huge application potential of a food biotechnology in the field of nutrition and health is shown.

Description

Technical Field

[0001] The present invention belongs to the field of food biotechnology and relates to a blackened jujube juice, and specifically to a method for preparing probiotic blackened jujube juice based on solid-liquid step-by-step green precision fermentation. Background Art

[0002] Jujube, the dried, mature fruit of the rhamnaceae plant, is rich in nutrients such as amino acids, dietary fiber, minerals, and bioactive substances like cyclic adenosine monophosphate. It boasts benefits such as tonifying qi, nourishing blood, calming the mind, strengthening the spleen and stomach, and moistening the lungs. Traditional ways to consume red dates include eating them fresh, drying them, cooking them, and making soups. However, these methods have limitations in terms of taste, nutrient retention, and functional properties.

[0003] Blackened dates are a new type of processed red date product. They undergo a Maillard reaction and caramelization process under specific temperature and humidity conditions, without any additives. Compared to standard red dates, they contain higher levels of bioactive ingredients such as polysaccharides, triterpenoid acids, and polyphenols. Furthermore, their fruit aroma is enriched, resulting in improved quality.

[0004] Probiotic fermentation technology has been widely used in the food industry in recent years. Probiotics possess a variety of beneficial physiological functions, such as regulating intestinal flora balance, enhancing immunity, and promoting nutrient absorption. Probiotic fermentation can transform some food ingredients into substances with higher nutritional value and bioactivity, while also improving the taste, flavor, and texture of foods.

[0005] Chinese patent CN119924428A uses Lactobacillus plantarum to ferment wolfberry puree and concentrated juice. Chinese patent CN119699393A uses Lactobacillus rhamnosus and Lactobacillus plantarum to ferment jujube pulp to produce low-sugar fermented red jujube pulp. These technologies involve adding probiotics directly after juicing the raw materials. There is no prior art record of using probiotics to ferment blackened jujube juice. Summary of the Invention

[0006] Addressing a research and development gap in the existing technology, the present invention provides a method for preparing probiotic blackened jujube juice based on green, precise fermentation using a solid-liquid, step-by-step process. By screening the effectiveness of different bacterial strains, the present invention significantly increases the viable bacterial count after fermentation. Based on this, a novel probiotic beverage has been developed, further addressing the growing demand in the functional health food and beverage industry for fruit and vegetable juices fermented with multiple probiotics.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows: The present invention provides a method for preparing probiotic blackened jujube juice based on solid-liquid step-by-step green precision fermentation, comprising the following steps: (1) Preparation of blackened jujube juice: fresh red jujubes are selected, washed, sealed with water, and solid-state fermented. The jujubes are crushed after pitting, extracted with water, filtered, and the soluble solid content is adjusted. The blackened jujube juice is sterilized and set aside. (2) Activation of bacterial strains: The probiotic strain is inoculated into the culture medium, and after subculture, the bacterial cells are collected by centrifugation. Subsequently, the precipitate is washed to obtain the activated bacterial strain; (3) Fermentation: inoculating the activated bacteria into the blackened jujube juice, and then fermenting to obtain probiotic fermented blackened jujube juice.

[0008] Preferably, in step (1), the ratio of red dates to water is 4-6 g: 1 mL; the solid-state fermentation conditions are solid-state fermentation at 65°C-70°C and humidity of 80%-85% for 130-150 h; the extraction conditions are adding water to the red dates after solid-state fermentation and soaking them at 75-85°C for 2-3 h, and the ratio of red dates to water is 1-2 g: 5 mL; the soluble solids content of the blackened date juice is 10-12 °Brix.

[0009] Preferably, in step (2), the specific steps of activating the strain are: inoculating the probiotic strains into MRS broth culture medium respectively, culturing at 37°C for 12 hours, subculturing for 2-3 generations, collecting the bacterial cells by centrifugation, and then washing the precipitate three times with sterile saline, adjusting the concentration of the bacterial suspension, and obtaining the activated strain.

[0010] Preferably, the probiotics are: Lactobacillus plantarum ( Lactiplantibacillus plantarum , LP121), Streptococcus thermophilus ( Streptococcus thermophilus , ST021) and Lactobacillus casei ( Lacticaseibacillus casei , LC023) or more; the concentration of the activated bacteria is 1×10 8 CFU / mL.

[0011] Preferably, in step (3), the inoculation amount of the activated bacteria in the blackened jujube juice is 1-5%.

[0012] Preferably, in step (3), the activated bacteria are composed of a Lactobacillus plantarum suspension and a Streptococcus thermophilus suspension in a volume ratio of 1:1.

[0013] Preferably, in step (3), the fermentation is carried out at a constant temperature of 33-41°C for 12-60 hours.

[0014] The beneficial effects of the present invention are: (1) The present invention achieves a synergistic growth effect by screening a specific combination of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus casei. After Lactobacillus plantarum and Streptococcus thermophilus are mixed in a 1:1 ratio, the number of viable cells at the end of fermentation is increased to ≥1×10 9 CFU / mL is significantly higher than single strain fermentation or other combined fermentation (2) The present invention screens and explores a probiotic compound combination that can effectively increase the number of live bacteria. By fermenting blackened jujube juice, the number of live probiotics in the product is increased while retaining the rich nutritional components of blackened jujube, thereby improving the functionality of the product.

[0015] (3) The present invention further reduces the sugar content in blackened jujube juice and increases the content of nutrients such as organic acids through precise fermentation of probiotics. Through the symbiotic metabolic regulation of the two bacteria, the sugar in the jujube juice is promoted to be efficiently converted into lactic acid (sugar degradation rate ≥ 25%). At the same time, key aroma substances such as ethyl acetate and n-hexanol are generated, giving the product a refreshing fruity aroma and low-sugar characteristics, thereby improving the flavor and taste of the beverage and making the blackened jujube juice have higher biological functionality. In addition, through the blood sugar-lowering effect of probiotics, a healthier beverage option is provided for people with low sugar needs, demonstrating the huge application potential of food biotechnology in the field of nutrition and health. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a single-factor experiment with different fermentation conditions (from left to right: inoculation amount, fermentation temperature, fermentation time) of the present invention; Figure 2 is the change of pH value and acidity during the fermentation process of the present invention; Figure 3 is the change of total sugar content during the fermentation process of the present invention; Figure 4 It is the change of lactic acid content during the fermentation process of the present invention. DETAILED DESCRIPTION

[0017] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0018] The probiotic Lactobacillus plantarum used in the present invention Lactiplantibacillus plantarum , LP121), Streptococcus thermophilus ( Streptococcus thermophilus , ST021) and Lactobacillus casei ( Lacticaseibacillus casei , LC023) was purchased from Weikaihaisi (Shandong) Bioengineering Co., Ltd.

[0019] Example 1 (1) Preparation of blackened jujube juice 1) Solid-state fermentation blackening treatment of raw materials Fresh red dates were selected, washed, and then watered in a sealed bag (600 g jujube:400 mL water ratio). Solid-state fermentation was performed at 65 ± 2°C and 80 ± 5% humidity for 130–150 h. After pitting, the dates were crushed and then extracted with water (1:5 w / v) at 75–85°C for 2 h. After filtration, the soluble solids content was adjusted to 10° Brix.

[0020] 2) Sterilization The jujube juice was sterilized in a water bath at 80°C for 25 min and rapidly cooled to 37°C.

[0021] (2) Bacteria selection and activation 1) Bacteria activation Select Lactobacillus plantarum ( Lactiplantibacillus plantarum , LP121), Streptococcus thermophilus ( Streptococcus thermophilus , ST021) and Lactobacillus casei ( Lacticaseibacillus casei , LC023). The three probiotic strains were inoculated into MRS broth and cultured at 37°C for 12 hours. After 2-3 subcultures to ensure activation, the cells were harvested by centrifugation at 3000-4000 rpm for 5 minutes. The pellet was then washed three times with sterile saline to remove residual culture medium components. Finally, the suspension concentration was adjusted to 10 using a McFarland standard turbidimetric tube. 8 CFU / mL 2) Bacterial strain selection Seven experimental groups were set up under sterile conditions: (a) single culture of Lactobacillus plantarum, (b) single culture of Streptococcus thermophilus, (c) single culture of Lactobacillus casei, (d) co-culture of Lactobacillus plantarum and Streptococcus thermophilus (1:1 volume ratio), (e) co-culture of Lactobacillus casei and Streptococcus thermophilus (1:1 volume ratio), (f) co-culture of Lactobacillus plantarum and Lactobacillus casei (1:1 volume ratio), and (g) triple culture of Lactobacillus plantarum, Streptococcus thermophilus, and Lactobacillus casei (1:1:1 volume ratio). The strains of the above seven experimental groups were inoculated into the same batch of blackened jujube juice at an inoculation ratio of 3%, and fermented under the same conditions. The number of viable bacteria was analyzed (the results are shown in Table 1). The co-culture of Lactobacillus plantarum and Streptococcus thermophilus in group d (1:1 volume ratio) had the best fermentation effect, with the number of viable bacteria reaching 1.59×10 9 CFU / mL, significantly higher than other experimental groups ( p <0.05), so Lactobacillus plantarum and Streptococcus thermophilus (1:1) were selected as the probiotic black date juice fermentation strain.

[0022] Table 1 Number of viable bacteria in fermentation combinations of different strains (3) Fermentation process optimization The single-factor optimization method was used to systematically investigate the probiotic fermentation process of jujube juice. The experimental design covers three key parameters: fermentation temperature (33°C, 35°C, 37°C, 38°C, 41°C), fermentation time (12h, 24h, 36h, 48h, 60h) and bacterial suspension inoculation amount (1%, 2%, 3%, 4%, 5%). The number of live bacteria in the fermentation product was used as the core evaluation index, and the fermentation strain was a mixed bacteria of plant lactobacillus and thermophilic streptococcus in a ratio of 1:1. Through the single-factor rotation test, it was found that when the inoculation amount was 3%, the optimal fermentation effect could be obtained by culturing the fermentation system at a constant temperature of 37°C for 48 hours. The results are shown in the figure. Figure 1 At this time, the number of viable bacteria reached a peak (1.57×10 9 CFU / mL), significantly higher than those in other experimental groups ( p <0.05).

[0023] Example 2 (1) Preparation of blackened jujube juice 1) Solid-state fermentation blackening treatment of raw materials Fresh red dates were selected, washed, and then watered in a sealed bag (600 g jujube:400 mL water ratio). Solid-state fermentation was performed at 65°C and 80% humidity for 144 hours. After pitting, the dates were crushed and then extracted with water (1:5 w / v) at 75°C for 2 hours. After filtration, the soluble solids content was adjusted to 10° Brix.

[0024] 2) Sterilization The jujube juice was sterilized in a water bath at 80°C for 25 min and then cooled to 37°C.

[0025] (2) Bacteria activation 1) Bacteria activation The probiotic strains were inoculated into MRS broth and cultured at 37°C for 12 hours. After 2-3 generations of subculture to ensure activation, the bacterial cells were collected by centrifugation at 3000-4000 rpm for 5 minutes. Subsequently, the precipitate was washed three times with sterile saline to remove residual culture medium components. Finally, the concentration of the bacterial suspension was adjusted to 10 using a McFarland standard turbidimetric tube. 8 CFU / mL (3) Fermentation The Lactobacillus plantarum suspension and the Streptococcus thermophilus suspension were inoculated into the blackened jujube juice at a ratio of 1:1, and cultured at a constant temperature of 37°C for 48 h to obtain the probiotic fermented blackened jujube juice.

[0026] Effect embodiment The probiotic fermented blackened jujube juice prepared in Example 2 was subjected to relevant tests, and the specific results were as follows: (1) Determination of pH and acidity Activated Lactobacillus plantarum and Streptococcus thermophilus were inoculated into sterilized, cooled blackened jujube juice for fermentation. The pH of the juice was measured during the fermentation process. Besides pH changes, acidity is also an important indicator during the fermentation process, reflecting the total amount of acidic substances produced by probiotic fermentation. Acidity was determined using an acid-base titration method in accordance with GB12456-2021. The total acid content of the product was also measured during the fermentation process.

[0027] like Figure 2 As shown, fermentation caused the pH to continuously decrease from 3.74 to 3.45. The total acid content increased from 3.88 mg / mL to 4.79 mg / mL. These data indicate that during the fermentation process, probiotics metabolize and produce acidic substances, causing the pH to decrease and the acidity to increase, affecting the flavor and health benefits of the final product.

[0028] (2) Determination of total sugar The polysaccharide content was determined by the phenol-sulfuric acid method. Accurately weigh 10 mg of anhydrous glucose dried to constant weight and prepare a 0.1 mg / mL glucose standard solution. Pipette 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of the standard solution into colorimetric tubes and add distilled water to 2.0 mL, add 2 mL of 50 g / L phenol solution, mix well, and then add 8 mL of concentrated sulfuric acid. Boil in a water bath for 20 min. Use a visible spectrophotometer and use the reagent blank solution as a blank control to measure the absorbance at a wavelength of 485 nm. Draw a standard curve with the mass concentration of glucose as the horizontal axis and the absorbance as the vertical axis. The regression equation is y = 0.2413x + 0.075, R 2 =0.997.

[0029] like Figure 3 As shown in the figure, the total sugar content continued to decrease from 2.48 mg / mL at 0 h to 1.80 mg / mL at 48 h, and the total sugar reduction effect reached 27.15%.

[0030] (3) Determination of lactic acid The content of lactic acid in fermented jujube juice was determined by high-performance liquid chromatography. A 20 mg / mL lactic acid standard solution was prepared and serially diluted with 0.1% aqueous phosphoric acid to concentrations of 3 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.25 mg / mL. Elution was performed using a mobile phase consisting of 0.1% aqueous phosphoric acid and methanol in a ratio of 97:3 at a flow rate of 1 mL / min. UV-visible detection was performed at 210 nm, with an injection volume of 10 μL and a column temperature of 40°C. A standard curve was constructed based on the peak areas of the standard solutions, and the lactic acid concentration in the samples was calculated based on the standard curve.

[0031] like Figure 4 As shown in the figure, the lactic acid content increased from 0.66 mg / mL to 1.58 mg / mL. The production of lactic acid can directly reflect the activity of the strain and the fermentation effect.

[0032] (4) Determination of volatile flavor substances Volatile flavor compounds were analyzed using headspace solid-phase microextraction (SPME)-gas chromatography-tandem mass spectrometry (GC-MS). 7.5 mL of probiotic-fermented blackened jujube juice was added to a 20 mL headspace vial. 1.0 g of NaCl and 10 μL of 2-octanol as an internal standard were added and dissolved thoroughly. After equilibration in a 40°C water bath for 30 minutes, a SPME fiber was inserted into the vial. After 30 minutes of adsorption, the fiber was immediately removed and inserted into the GC inlet for 5 minutes of desorption prior to GC-MS analysis. GC conditions included a DB-5MS (30 m × 0.25 mm, 0.25 μm) column with split injection (split ratio 10:1). Column temperature: 40°C for 5 minutes, then heated to 100°C at a rate of 8°C / min, then to 220°C at a rate of 5°C / min, where it was held for 5 minutes. The carrier gas was He, the column pressure was 50 kPa, the total flow rate was 34 mL / min, the column flow rate was 1 mL / min, and the inlet temperature was 230 °C. MS conditions included an EI + ionization source with an electron energy of 70 eV, a detector voltage of 0.1 kV, and an ion source temperature of 230 °C. The calculation formula was: C = A × C i / A i .

[0033] Where C is the concentration of volatile substances, A is the chromatographic peak area of ​​volatile substances; C i is the concentration of internal standard 2-octanol, A i is the chromatographic peak area of ​​the internal standard 2-octanol.

[0034] The changes in flavor substances are shown in Table 2. The volatile flavor substances increased from 26 at 0 h to 30 at 48 h. Among them, n-hexanol, nonanol, heptanol, 2-acetylpyrrole, and ethyl acetate were generated, which gave the probiotic fermented jujube juice a unique flavor.

[0035] Table 2 Changes in volatile flavor characteristics during fermentation NF means not detected.

Claims

1. A method for preparing probiotic blackened jujube juice based on solid-liquid step-by-step green precision fermentation, characterized in that: The following steps are involved: (1) Preparation of blackened jujube juice: fresh red jujubes are selected, washed, sealed with water, and solid-state fermented. The jujubes are crushed after pitting, extracted with water, filtered, and the soluble solid content is adjusted. The blackened jujube juice is sterilized and set aside. (2) Activation of bacterial strains: The probiotic strain is inoculated into the culture medium, and after subculture, the bacterial cells are collected by centrifugation. Subsequently, the precipitate is washed to obtain the activated bacterial strain; (3) Fermentation: inoculating the activated bacteria into the blackened jujube juice, and then fermenting to obtain probiotic fermented blackened jujube juice.

2. The method according to claim 1, characterized in that In step (1), the ratio of red dates to water is 4-6 g: 1 mL; the solid-state fermentation conditions are 65°C-70°C and 80%-85% humidity for 130-150 h; the extraction conditions are adding water to the red dates after solid-state fermentation and soaking them at 75-85°C for 2-3 h, and the ratio of red dates to water is 1-2 g: 5 mL; the soluble solids content of the blackened date juice is 10-12 °Brix.

3. The method according to claim 1 or 2, characterized in that In step (2), the specific steps of activating the strains are as follows: the probiotic strains are inoculated into MRS broth culture medium respectively, cultured at 37°C for 12 hours, subcultured for 2-3 generations, and the bacterial cells are collected by centrifugation. Subsequently, the precipitate is washed three times with sterile saline, and the concentration of the bacterial suspension is adjusted to obtain the activated strains.

4. The method according to claim 3, characterized in that The probiotics are: Lactobacillus plantarum ( Lactiplantibacillus plantarum , LP121), Streptococcus thermophilus ( Streptococcus thermophilus , ST021) and Lactobacillus casei ( Lacticaseibacillus casei , LC023) or more; the concentration of the activated bacteria is 1×10 8 CFU / mL.

5. The method according to claim 1, 3 or 4, characterized in that In step (3), the inoculation amount of the activated bacteria in the blackened jujube juice is 1-5%.

6. The method according to claim 1, 3 or 4, characterized in that In step (3), the activated bacteria are composed of a Lactobacillus plantarum suspension and a Streptococcus thermophilus suspension in a volume ratio of 1:

1.

7. The method according to claim 1 or 6, characterized in that In step (3), the fermentation is carried out at a constant temperature of 33-41°C for 12-60 hours.

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