A probiotic-based fermented citrus beverage and its preparation process

Abstract

This invention belongs to the field of fermentation technology, specifically relating to a probiotic-based fermented citrus beverage and its preparation process. The preparation process of the probiotic-based fermented citrus beverage includes the following steps: S1, pretreatment of raw juice to obtain fermentation base liquid; S2, inoculation; S3, fermentation to obtain fermentation broth; S4, post-treatment. By using a compound enzyme preparation to directionally degrade bitter substances, employing a compound acid-resistant bacterial strain for synergistic fermentation, and combining substrate optimization, staged temperature-controlled fermentation, and low-temperature post-treatment technology, the content of naringin and limonene is significantly reduced, while the retention rates of total phenols and total flavonoids and the survival rate of probiotics are improved. This results in a citrus beverage with excellent flavor, nutritional and health benefits, and stability.

Description

Technical Field

[0001] This invention belongs to the field of fermentation technology, specifically relating to a probiotic-based fermented citrus beverage and its preparation process. Background Technology

[0002] Probiotic drinks are widely popular due to their health benefits, such as regulating gut microbiota and boosting immunity. Currently, most probiotic drinks on the market are based on dairy products like yogurt and lactic acid bacteria drinks, or fermented grains. Citrus drinks are favored by consumers for their high vitamin C and bioactive components, but current preparation processes have many problems.

[0003] Bitter substances such as naringin and limonene in citrus fruits are difficult to remove effectively, and traditional single enzymatic hydrolysis or chemical debittering methods easily destroy nutrients. Probiotics have a low survival rate in the acidic environment of citrus fruits, and high-temperature processing and storage further lead to a sharp drop in the number of live bacteria, making it difficult to achieve sustained health benefits. At the same time, high-temperature sterilization and rough processing in traditional processes result in the significant loss of heat-sensitive components such as vitamin C, making the product prone to sedimentation and stratification, and exhibiting poor stability. Furthermore, existing products mostly rely on single-strain fermentation, resulting in limited functionality and insufficient flavor harmony, failing to meet consumers' demand for high-quality, multifunctional health drinks.

[0004] Therefore, it is of great significance to develop a citrus beverage preparation process that can efficiently remove bitterness, achieve high probiotic survival rate, retain nutrients well, and maintain excellent stability. Summary of the Invention

[0005] The purpose of this invention is to provide a probiotic-fermented citrus beverage and its preparation process, which significantly reduces the content of naringin and limonene, increases the retention rate of total phenols and total flavonoids and the survival rate of probiotics, so that the prepared citrus beverage has excellent flavor, nutritional and health value and stability.

[0006] A preparation process for a probiotic-fermented citrus beverage includes the following steps:

[0007] S1. Pretreatment of raw juice to obtain fermentation base liquid;

[0008] S2, Inoculation;

[0009] S3. Fermentation to obtain fermentation liquid;

[0010] S4, Post-processing.

[0011] Preferably, in step S1, the specific steps of raw juice pretreatment are as follows: juicing citrus, passing the juice through an 80-150 mesh sieve to obtain citrus raw juice, adding enzyme preparation for enzymatic hydrolysis, adding sugar source and stirring evenly, adjusting the pH to 4.2-4.8, pasteurizing, and cooling to 30-32℃.

[0012] Preferably, the enzyme preparation includes naringinase, β-glucosidase, cellulase, and neutral protease.

[0013] Preferably, the enzyme preparation is added at a concentration of 130-200 U / mL of citrus juice.

[0014] Preferably, the ratio of the amount of naringinase, β-glucosidase, cellulase and neutral protease added is (3-5):(1-3):(1-2):1.

[0015] By selecting a specific blend of enzymes, bitter substances can be targeted for degradation, improving the release efficiency of active nutritional components and enhancing flavor and mouthfeel. This is likely because, on the one hand, the synergistic action of naringinase and β-glucosidase selectively breaks down the molecular structure of bitter substances, reducing their content at the source. On the other hand, cellulase disrupts the cell wall structure of citrus fruits, and neutral protease decomposes proteins bound to pectin. These two enzymes work together to disrupt cell structure, promoting the release of fat-soluble active components such as total phenols and total flavonoids, reducing the interference of protein impurities on flavor. By optimizing the blending ratio of the four enzymes, the content of naringin and limonene in beverages can be significantly reduced, while the content of total phenols and total flavonoids can be increased, improving the basic flavor base.

[0016] Preferably, the specific steps of the enzymatic hydrolysis are as follows: first, add naringinase, β-glucosidase and cellulase, and hydrolyze for 1-2 hours at a pH of 4.0 and a temperature of 40-50℃, with a stirring speed of 40-60 rpm. Then, adjust the pH to 6.5-7.5, add neutral protease, and keep the temperature and stirring speed constant for 45-60 minutes.

[0017] Preferably, the sugar source is fructooligosaccharide or inulin.

[0018] Preferably, the amount of sugar source added is 2%-6% of the mass of the citrus juice.

[0019] Preferably, the pasteurization conditions are: a temperature of 85-90℃ and a time of 1-3 minutes.

[0020] Preferably, in step S2, the specific step of inoculation is: inoculating the fermentation base liquid with a compound probiotic fermentation agent.

[0021] Preferably, the compound probiotic fermentation agent includes Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus.

[0022] Preferably, the inoculum size of the compound probiotic starter is 1×10⁻⁶. 7 -5×10 7 CFU / mL fermentation broth

[0023] Preferably, the inoculation ratio of Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus is (4-6):(3-5):1; more preferably, it is 5:4:1.

[0024] By selecting specific probiotic strains as fermentation agents, the survival stability of probiotics can be enhanced, thus strengthening the nutritional and health benefits of citrus beverages. This is likely because all three bacteria can survive under acidic conditions, forming a synergistic acid-resistant system. The organic acids produced by *Lactobacillus plantarum* metabolism maintain pH stability, providing a suitable growth environment for *Lactobacillus rhamnosus* and *Lactobacillus acidophilus*. The extracellular polysaccharides secreted by *Lactobacillus rhamnosus* can encapsulate the bacterial cells, reducing the damage to cell structure caused by acidic conditions. *Lactobacillus acidophilus* inhibits the growth of other bacteria through nutrient competition. By optimizing the ratio of the three probiotic strains and improving the intertrophic effect, the survival stability of probiotics during fermentation and storage is significantly improved. Furthermore, the acid-resistant strains reduce vitamin C oxidation at low pH, thereby increasing vitamin C retention. In addition, the antioxidant enzymes produced by the bacteria's metabolism protect phenolic substances from oxidation, improving the stability of total phenols, total flavonoids, and other nutrients and reducing their loss, thus ensuring intestinal regulatory function.

[0025] Preferably, in step S3, the fermentation is a two-stage temperature-controlled fermentation. The specific fermentation steps are as follows: in the first stage, micro-aerobic fermentation is carried out at 30-32℃ for 18-24 hours. When the pH drops to 3.8-4.2, oligofructose is added, the temperature is lowered to 20-25℃, and the process is transferred to the second stage, anaerobic fermentation is carried out at 20-25℃ for 12-24 hours. The process is then rapidly cooled to 4-10℃ to terminate the fermentation.

[0026] Preferably, the dissolved oxygen concentration during microaerobic fermentation is controlled at 0.2-0.4 mg / L.

[0027] Preferably, the amount of fructooligosaccharide added is 3%-5% of the mass of the fermentation base liquid.

[0028] Two-stage temperature-controlled fermentation improves fermentation efficiency, directionally regulates the accumulation of flavor compounds and active ingredients, enhances taste harmony, and increases the content of total phenols and flavonoids. This is because in the first stage, *Lactobacillus plantarum* and *Lactobacillus acidophilus* proliferate rapidly, producing large amounts of acid and initiating the degradation of bitter substances. When the pH drops to 3.8-4.2, supplementing with fructooligosaccharides prevents the bacteria from entering a period of decline due to insufficient carbon source. Then, the second stage, conducted at low temperatures, inhibits the rate of acid production, reducing astringency caused by excessive acidification. Simultaneously, *Lactobacillus rhamnosus* synthesizes esters and other flavor compounds more readily at low temperatures, imparting a refreshing aroma to the beverage. Furthermore, increased cell membrane permeability at low temperatures promotes the dissolution of phenols and flavonoids from citrus fruits. This staged temperature control achieves the orderly progression of "efficient debittering - flavor harmonization - active ingredient accumulation."

[0029] Preferably, in step S4, the specific post-processing steps are as follows: after centrifuging the fermentation broth at low temperature, collect the centrifuged liquid, add a natural stabilizer, stir evenly, filter aseptically through a 0.22μm membrane, pasteurize at low temperature, rapidly cool to 2-8℃, and refrigerate to obtain the citrus beverage.

[0030] Preferably, the specific conditions for the low-temperature centrifugation are: temperature of 4-10℃, rotation speed of 3000-5000 r / min, and time of 10-20 min.

[0031] By employing a gentle post-processing technique, probiotic loss is minimized, probiotic activity is preserved, centrifugal sedimentation rate is reduced, and product stability is improved. Low-temperature centrifugation removes large particulate impurities while avoiding heat damage to probiotics. High-fat pectin and gellan gum are selected as natural stabilizers. High-fat pectin binds with calcium ions in citrus to enhance gel strength, while gellan gum increases system viscosity. Together, they form a three-dimensional gel network that encapsulates tiny particles, reducing sedimentation. Low-temperature pasteurization inactivates contaminating bacteria while maximizing the preservation of probiotic activity. The gentle post-processing technique maintains low temperatures throughout the entire process, from physical separation to sterilization, achieving a dual guarantee of probiotic activity and product stability.

[0032] Preferably, the natural stabilizer includes one or more of high-ester pectin and gellan gum.

[0033] Preferably, the mass ratio of the high-fat pectin to gellan gum is (7-9):1.

[0034] Preferably, the amount of the natural stabilizer added is 0.1%-0.3% of the mass of the centrifuged liquid.

[0035] Preferably, the specific conditions for the low-temperature pasteurization are: a temperature of 60-65℃ and a time of 10-20s.

[0036] Through enzymatic hydrolysis, fermentation, and post-treatment, multiple stages are linked to achieve synergistic optimization of the flavor, nutrition, and stability of citrus beverages. The efficient debittering effect of the compound enzyme preparation lays a solid flavor foundation for subsequent fermentation; the compound acid-resistant bacteria proliferate efficiently under optimized substrate and fermentation conditions, and their metabolites further aid in the enzymatic hydrolysis of residual bitter substances; staged fermentation coordinates the acid production rate with the accumulation of flavor compounds; and gentle post-treatment preserves the activity of probiotics while fixing nutritional components with stabilizers. Each stage forms a closed-loop synergistic effect of "debittering-proliferation-preservation-stabilization," ultimately achieving a comprehensive improvement in the product's flavor, nutrition, and stability.

[0037] The citrus beverage prepared by the probiotic fermentation process is described above.

[0038] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0039] 1. This invention provides a probiotic-based fermented citrus beverage. It utilizes a compound enzyme preparation to target and degrade bitter substances, employs a compound acid-resistant bacterial strain for synergistic fermentation, and combines substrate optimization, staged temperature-controlled fermentation, and low-temperature post-treatment technology to significantly reduce the content of naringin and limonene, while increasing the retention rate of total phenols and total flavonoids and the survival rate of probiotics. This results in a citrus beverage that possesses excellent flavor, nutritional and health benefits, and stability.

[0040] 2. This invention, by selecting several specific enzymes in combination, can target and degrade bitter substances, improve the release efficiency of nutritional active ingredients, and enhance flavor and taste.

[0041] 3. By selecting specific probiotic strains as fermenting agents, this invention can enhance the survival stability of probiotics and strengthen the nutritional and health benefits of citrus beverages.

[0042] 4. This invention improves fermentation efficiency, regulates the accumulation of flavor substances and active ingredients, enhances taste harmony, and increases the content of total phenols and total flavonoids through two-stage temperature-controlled fermentation.

[0043] 5. This invention reduces probiotic loss and ensures probiotic activity through a gentle post-processing technique, thereby reducing centrifugal sedimentation rate and improving product stability. Detailed Implementation

[0044] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] All raw materials used in this invention are commercially available, as detailed below:

[0046] Naringinase, activity approximately 100 U / mg; β-glucosidase, activity approximately 100 U / g; cellulase, activity approximately 50 U / mg; neutral protease, activity approximately 100 U / mg; fructooligosaccharides; inulin; gellan gum; all from Shanghai Maclean Reagents.

[0047] High-fat pectin, from Jiangsu Yuanzhiyuan Biotechnology Co., Ltd.

[0048] Lactobacillus plantarum, strain number CICC 25125; Lactobacillus rhamnosus, strain number CICC 6137; Lactobacillus acidophilus, strain number CICC 6091; all were obtained from the China Industrial Microbial Culture Collection Center.

[0049] Example 1

[0050] This embodiment provides a preparation process for a probiotic-fermented citrus beverage, the steps of which are as follows:

[0051] S1. Pretreatment of raw juice to obtain fermentation base liquid;

[0052] S2, Inoculation;

[0053] S3. Fermentation to obtain fermentation liquid;

[0054] S4, Post-processing.

[0055] In step S1, the specific steps of raw juice pretreatment are as follows: juicing citrus, passing the juice through a 100-mesh sieve to obtain citrus raw juice, adding enzyme preparation for enzymatic hydrolysis, adding sugar source and stirring evenly, adjusting pH to 4.5, pasteurizing, and cooling to 30°C.

[0056] The enzyme preparation was added at a rate of 170 U / mL of citrus juice.

[0057] The enzyme preparation consists of naringinase, β-glucosidase, cellulase, and neutral protease, with an addition ratio of 4:2:1.5:1.

[0058] The specific steps and conditions for the enzymatic hydrolysis are as follows: first, add naringinase, β-glucosidase, and cellulase, and hydrolyze for 1.5 hours at a pH of 4.0 and a temperature of 45°C, with a stirring speed of 50 rpm. Then, adjust the pH to 7, add neutral protease, and keep the temperature and stirring speed constant for 50 minutes.

[0059] The sugar source is fructooligosaccharide.

[0060] The amount of sugar source added is 4% of the mass of the citrus juice.

[0061] The specific conditions for pasteurization are: temperature 85℃ and time 2 minutes.

[0062] In step S2, the specific steps of inoculation are: inoculating the fermentation base liquid with compound probiotic fermentation agent.

[0063] The compound probiotic starter consists of Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus, with an inoculation ratio of 5:4:1.

[0064] The inoculation amount of the compound probiotic starter is 2.5 × 10⁻⁶. 7 CFU / mL fermentation broth

[0065] In step S3, the fermentation is a two-stage temperature-controlled fermentation. The specific fermentation steps are as follows: in the first stage, micro-aerobic fermentation is carried out at 30°C for 20 hours. When the pH drops to 4.0, oligofructose is added, the temperature is lowered to 22°C, and the process is transferred to the second stage, where anaerobic fermentation is carried out at 22°C for 18 hours. The process is then rapidly cooled to 4°C to terminate the fermentation.

[0066] The dissolved oxygen concentration during microaerobic fermentation is controlled at 0.3 mg / L.

[0067] The amount of fructooligosaccharides added is 4% of the mass of the fermentation broth.

[0068] In step S4, the specific post-processing steps are as follows: after centrifuging the fermentation broth at low temperature, collect the centrifuged liquid, add a natural stabilizer, stir evenly, filter aseptically through a 0.22μm membrane, pasteurize at low temperature, rapidly cool to 4℃, and refrigerate to obtain the citrus beverage.

[0069] The specific conditions for the low-temperature centrifugation are: temperature 4℃, rotation speed 4000r / min, and time 15min.

[0070] The natural stabilizer is high-ester pectin and gellan gum in a mass ratio of 8:1.

[0071] The amount of the natural stabilizer added is 0.2% of the mass of the centrifuged liquid.

[0072] The specific conditions for low-temperature pasteurization are: temperature 60℃ and time 15s.

[0073] Example 2

[0074] The difference between this embodiment and Example 1 is that the enzyme preparation consists of naringinase, β-glucosidase, cellulase, and neutral protease, with an addition ratio of 4:3:1:1.

[0075] Comparative Example 1

[0076] The difference between this comparative example and Example 1 is that the enzyme preparation is naringinase, cellulase, and neutral protease, and the ratio of the added amounts is 4:1.5:1.

[0077] Comparative Example 2

[0078] The difference between this comparative example and Example 1 is that the enzyme preparations are naringinase, β-glucosidase, cellulase, and neutral protease, and the ratio of the added amounts is 7:1:1:1.

[0079] Comparative Example 3

[0080] The difference between this comparative example and Example 1 is that the enzyme preparation is naringinase.

[0081] Comparative Example 4

[0082] The difference between this comparative example and Example 1 is that the compound probiotic fermenting agent is Lactobacillus plantarum.

[0083] Comparative Example 5

[0084] The difference between this comparative example and Example 1 is that the compound probiotic starter is Lactobacillus plantarum and Lactobacillus rhamnosus, and the inoculation ratio is 5:4.

[0085] Comparative Example 6

[0086] The difference between this comparative example and Example 1 is that the compound probiotic starter is Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus, with an inoculation ratio of 8:1:1.

[0087] Comparative Example 7

[0088] The difference between this comparative example and Example 1 is that in step S3, the specific fermentation steps are as follows: in the first stage, fermentation is carried out at 30°C under micro-aerobic conditions for 38 hours, followed by rapid cooling to 4°C to terminate the fermentation.

[0089] Comparative Example 8

[0090] The difference between this comparative example and Example 1 is that in step S4, the specific post-processing steps are as follows: after centrifuging the fermentation broth at low temperature, collect the centrifuged liquid, add a natural stabilizer, stir evenly, aseptically filter through a 0.22μm membrane, pasteurize, rapidly cool to 4℃, and refrigerate to obtain the citrus beverage.

[0091] The specific conditions for pasteurization are: temperature 85℃ and time 30s.

[0092] Performance testing

[0093] The method described in patent CN 108308481B was used to test the polyphenol content in citrus beverages. Simultaneously, the contents of naringin and limonene in both citrus juice and beverages were tested. The removal rates of naringin and limonene were calculated using the formula: (Content of a substance in citrus juice - Content of a substance in citrus beverage) / Content of a substance in citrus juice × 100%. The total flavonoid content in citrus beverages was tested according to GB / T20574-2006. The vitamin C content in both citrus juice and beverages was tested according to GB 5009-2016, and the vitamin C retention rate was calculated as: (Vitamin C content in citrus beverage / Vitamin C content in citrus juice) × 100%. The number of live probiotics in citrus beverages was tested according to GB 4789-2016. Stability test: Take 10 mL of sample, centrifuge at 4000 r / min for 20 min at 4℃, discard the supernatant, and dry the precipitate at 105℃ to constant weight. Calculate the precipitation rate = (precipitate mass / sample mass) × 100%. The results are shown in Table 1.

[0094] Table 1 Measurement Results

[0095]

[0096] According to statistics, the probiotic-fermented citrus beverages prepared in Examples 1-2 of this invention have high contents of total phenols and total flavonoids, high viable probiotic counts, and high vitamin C retention rates, indicating that the prepared citrus beverages have excellent nutritional and health benefits. Furthermore, the removal rates of bitter substances such as naringin and limonene are high, contributing to flavor enhancement. In addition, the low precipitation rate in the centrifugation sedimentation test indicates high product stability. Comparative Example 1 lacked β-glucosidase; Comparative Example 2 had an imbalanced enzyme ratio; Comparative Example 3 was treated with only naringin; Comparative Example 4 used only *Lactobacillus plantarum*; Comparative Example 5 lacked *Lactobacillus acidophilus*; Comparative Example 6 had an imbalanced ratio of compound probiotic starter; Comparative Example 7 used one-stage constant temperature fermentation; Comparative Example 8 used high-temperature pasteurization in step S4. The resulting citrus beverages had low contents of total phenols, total flavonoids, and probiotics, low vitamin C retention rates, poor nutritional value, low removal rates of naringin and limonene, a bitter taste, and low centrifugation sedimentation rates. This indicates that the citrus beverage prepared using the raw materials and methods described in this application possesses excellent flavor, nutritional and health benefits, and stability.

[0097] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A preparation process for a probiotic-fermented citrus beverage, characterized in that, Includes the following steps: S1. Juice the citrus fruits, sieve to obtain the original citrus juice, add enzyme preparation for enzymatic hydrolysis, add sugar source and stir evenly, adjust pH, pasteurize, cool to obtain fermentation base liquid; the enzyme preparation includes naringinase, β-glucosidase, cellulase and neutral protease, and the ratio of the added amounts is (3-5):(1-3):(1-2):

1. The specific steps of enzymatic hydrolysis are as follows: First, add naringinase, β-glucosidase, and cellulase. Under the conditions of pH 4.0 and temperature of 40-50℃, the enzymatic hydrolysis time is 1-2 hours, the stirring speed is 40-60 rpm, and the pH is adjusted to 6.5-7.

5. Then, add neutral protease, keep the temperature and stirring speed constant, and enzymatically hydrolyze for 45-60 minutes. S2. Inoculate the fermentation base liquid with a compound probiotic starter; the compound probiotic starter includes Lactobacillus plantarum, Lactobacillus rhamnosus, and Lactobacillus acidophilus, and the ratio of the inoculation amounts is (4-6):(3-5):1; S3. Fermentation: In the first stage, micro-aerobic fermentation is carried out at 30-32℃ for 18-24 hours. When the pH drops to 3.8-4.2, oligofructose is added, and the temperature is lowered to 20-25℃ to enter the second stage, anaerobic fermentation at 20-25℃ for 12-24 hours. The temperature is then rapidly cooled to 4-10℃ to terminate the fermentation and obtain the fermentation broth. S4. After centrifuging the fermentation broth at low temperature, collect the centrifuged liquid, add a natural stabilizer, stir evenly, and then aseptically filter through a 0.22μm filter membrane. Perform low-temperature pasteurization, rapidly cool to 2-8℃, and refrigerate to obtain the citrus beverage. The specific conditions for low-temperature pasteurization are: temperature 60-65℃ and time 10-20s.

2. The preparation process of the probiotic-fermented citrus beverage according to claim 1, characterized in that, The enzyme preparation is added at a rate of 130-200 U / mL of citrus juice.

3. The preparation process of the probiotic-fermented citrus beverage according to claim 2, characterized in that, The inoculation amount of the compound probiotic fermentation agent is 1×10⁻⁶. 7 -5×10 7 CFU / mL fermentation broth 4. A citrus beverage prepared according to any one of claims 1 to 3 based on the probiotic fermentation process for citrus beverages.

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