Preparation method and application of probiotic fermented apple juice

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method and application of probiotic fermented apple juice. The probiotic is Kluyveromyces marxianus CICC 32017, and the Kluyveromyces marxianus can overcome the defects that the fruit and vegetable juice is insufficient in fragrance and heavy in raw and green taste when being singly fermented by lactic acid bacteria. The method for preparing the fermented apple juice comprises the following steps: juicing apples; performing enzymolysis; performing sterilization; the method comprises the following steps of: inoculating the apple juice with a Kluyveromyces marxianus CICC 32017 or a mixed microbial agent of the Kluyveromyces marxianus CICC 32017 and lactobacillus plantarum CICC 21801, and culturing for 48-72 hours in a constant-temperature incubator at the temperature of 30 DEG C, so that a large amount of organic acid is generated after fermentation is finished, the DPPH free radical scavenging rate and the ABTS free radical scavenging rate of the fermented apple juice are higher than those of a control group, the oxidation resistance is remarkably improved, meanwhile, aroma substances are increased, and the taste of the apple juice is improved. The quality of the fermented apple juice is greatly improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to a preparation method and application of probiotic fermented apple juice. Background Art

[0002] Apple belongs to the Rosaceae family and is rich in sugar, acid, vitamins, flavonoids, dietary fiber, pectin, mineral elements and other nutrients, showing extremely high nutritional value. Its sweet substance sugar can please the body and mind, while acid has multiple functions such as eliminating fatigue, increasing appetite, promoting digestion, softening blood vessels and lowering blood cholesterol. Especially malic acid and citric acid can also promote the body's absorption of calcium. Vitamin C plays an important role in enhancing immunity, preventing anemia, promoting collagen formation, maintaining bone and tooth function and cell membrane integrity, detoxification and anti-aging. Flavonoids, as key polyphenols in apples, have biological activities such as antibacterial, antiviral, anti-inflammatory, anti-allergic and inhibition of various biological enzyme activities. In addition, the dietary fiber in apples has a significant effect on preventing and treating obesity, diabetes, hypertension, and preventing coronary heart disease, arteriosclerosis, constipation and gallstones.

[0003] Kluyveromyces marxii is a multifunctional fungal microorganism with good heat resistance and the ability to utilize a variety of carbon sources. It can produce beneficial short-chain fatty acids and a variety of antioxidant metabolites, and has the functions of anti-oxidation, regulating intestinal flora, and promoting the growth of beneficial flora. Kluyveromyces marxii can produce a variety of alcohols, esters, and aldehydes, and its ethanol production capacity is relatively weak. It has the effect of improving the aroma quality and functional characteristics of products in food fermentation. In addition, Kluyveromyces marxii has good acid resistance, and mixed fermentation with acid-producing plant lactic acid bacteria to compete for nutrients can avoid excessive fermentation of plant bacteria and improve the shortcomings of insufficient aroma and heavy green taste of fruit and vegetable juice fermented by lactic acid bacteria alone. Therefore, the application of Kluyveromyces marxii in apple juice fermentation has broad development prospects. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a preparation method and application of probiotic fermented apple juice.

[0005] The first object of the present invention is to provide a strain of Kluyveromyces marxii CICC 32017, which has a strong ability to improve the aroma of fermented fruit and vegetable juice.

[0006] The colony of the Kluyveromyces marxii yeast of the present invention is round, white, and has a smooth surface. It is in a protruding state on the surface of the solid culture medium with neat and smooth edges, and grows well in a relatively high acidity environment.

[0007] The second object of the present invention is to provide a strain combination, comprising the Kluyveromyces marxianus CICC32017 and Lactobacillus plantarum CICC 21801.

[0008] The third object of the present invention is to provide a method for preparing probiotic fermented apple juice, comprising the following steps:

[0009] (1) Apple juice;

[0010] (2) Enzymatic hydrolysis;

[0011] (3) Sterilization;

[0012] (4) preparing bacterial agents;

[0013] (5) Inoculation and fermentation: Activated Kluyveromyces marxii CICC 32017, or Kluyveromyces marxii CICC 32017 and Lactobacillus plantarum CICC 21801 are inoculated into the apple juice prepared in step (3) for fermentation.

[0014] The enzymatic hydrolysis method in step (2) is to add pectinase and cellulase in a mass ratio of 1-3:1-3, and the total amount of enzyme added is 0.2-0.5% of the mass of the apple juice. The enzymatic hydrolysis is carried out at 45-50° C. for 3-5 hours, and the apple juice is filtered to obtain the apple juice.

[0015] The sterilization temperature in step (3) is 80-100°C and the time is 10-20 minutes.

[0016] The preparation methods of the Kluyveromyces marxkei (CICC 32017) and Lactobacillus plantarum (CICC21801) bacterial agents described in step (4) are as follows: streaking a Kluyveromyces marxkei (CICC 32017) bacterial solution preserved in 50% glycerol at -80°C on a YPD agar medium, culturing at 25-30°C for 48-72h to obtain a single colony, picking a single colony with an inoculation loop and placing it in a YPD liquid medium, culturing at 25-30°C for 48-72h to obtain a propagation culture solution, centrifuging the bacterial solution to collect the bacterial bodies, washing them twice with sterile water, and freeze-drying them in a freeze dryer to obtain freeze-dried bacterial powder. The bacterial liquid of Lactobacillus plantarum (CICC21801) preserved in 50% glycerol at -80°C was streaked on an MRS agar medium, and cultured at 30-37°C for 24-48 hours to obtain a single colony. The single colony was picked up with an inoculation loop and placed in an MRS broth medium, and cultured at 30-37°C for 24-48 hours to obtain the bacterial bodies by centrifugation, washed twice with sterile water, and freeze-dried in a freeze dryer to obtain freeze-dried bacterial powder.

[0017] The inoculation amount in step (5) is 0.1-0.4 g bacterial powder / 100 g juice. When Kluyveromyces marxianus CICC 32017 and Lactobacillus plantarum CICC 21801 are mixed for use, the mass ratio of Kluyveromyces marxianus to Lactobacillus plantarum in the mixed bacterial fermentation is 1-2:1-2.

[0018] The fermentation conditions in step (5) are culturing in a constant temperature incubator at 25 to 30° C. for 48 to 72 hours.

[0019] The fourth object of the present invention is to provide apple juice prepared by the method.

[0020] The fifth object of the present invention is to provide an application of apple juice in preparing antioxidant products.

[0021] Beneficial effects of the present invention: The Kluyveromyces marxii yeast of the present invention has a faster growth rate and a stronger ability to produce aroma, and a weaker ability to produce ethanol, and can be well applied to fruit juice fermentation to improve product quality. The embodiment of the present invention specifically provides the application of the bacteria mixed with plant lactobacillus in fermenting apple juice. The yeast has good acid resistance, and competes with plant lactobacillus for the utilization of nutrients in apple juice, and can inhibit the growth of plant lactobacillus to a certain extent, and can avoid excessive growth and acid production of plant lactobacillus, and improves the shortcomings of insufficient aroma and heavy green taste of fruit and vegetable juice fermented by lactic acid bacteria alone by producing aroma components and beneficial metabolites, and increases the antioxidant activity of the product. The DPPH and ABTS free radical scavenging rates of apple juice after fermentation by the present invention are higher than those of the control group, and the antioxidant activity is significantly increased. At the same time, the aroma substances are increased, so that the quality of the fermented apple juice is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a colony of Kluyveromyces marxii CICC 32017 of the present invention;

[0023] Figure 2 is the pH of the fermented apple juice according to different embodiments of the present invention;

[0024] Figure 3 is the DPPH radical scavenging capacity of the fermented apple juice according to different embodiments of the present invention;

[0025] Figure 4 is the ABTS free radical scavenging capacity of the apple juice fermented according to different embodiments of the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. Specific conditions are not specified in the embodiments, and the conditions recommended by the manufacturer are carried out according to conventional conditions or manufacturers. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0027] The Kluyveromyces marxii (CICC 32017) and Lactobacillus plantarum (CICC 21801) of the present invention were purchased from the China Industrial Microbiological Culture Collection Administration Center.

[0028] Example 1

[0029] The Kluyveromyces marxianus CICC 32017 of the present invention can effectively improve the shortcoming of insufficient aroma of single fermentation of Lactobacillus plantarum, and the yeast is acid-resistant and has a fast growth rate. The colony characteristics of Kluyveromyces marxianus CICC 32017 are as follows Figure 1 As shown, the colony features are round, white, and smooth, and are protruding on the surface of the solid culture medium with neat and smooth edges. A method for preparing fermented apple juice comprises the following steps:

[0030] (1) Apple juice extraction: Select undamaged and rotten apples, wash them, cut them into pieces and extract the juice.

[0031] (2) Enzymatic hydrolysis: pectinase and cellulase were added in a mass ratio of 3:2, and the total amount of enzyme added was 0.5 g / 100 g of apple juice. The enzymatic hydrolysis was performed at 50° C. for 4.2 h, and the apple juice was obtained by filtration.

[0032] (3) Sterilization: The apple juice after enzymatic filtration was sterilized by high pressure steam at 90°C for 10 min.

[0033] (4) Preparation of bacterial agent: A bacterial solution of Kluyveromyces marxii (CICC 32017) preserved in 50% glycerol at -80°C was streaked on YPD agar medium, and incubated at 30°C for 48 h to obtain a single colony. A single colony was picked up with an inoculation loop and placed in YPD liquid medium, and incubated at 30°C for 48 h to obtain a propagation bacterial solution. The bacterial solution was centrifuged to collect the cells, which were washed twice with sterile water and freeze-dried in a freeze dryer to obtain freeze-dried bacterial powder.

[0034] (5) Inoculation: Take the Kluyveromyces marxii powder and inoculate it into apple juice at a ratio of 0.3 g / 100 g juice, and ferment it at 30° C. for 48 h.

[0035] Example 2

[0036] The other conditions were the same as those in Example 1. The Kluyveromyces marxii and Lactobacillus plantarum powders were mixed evenly in a ratio of 2:1, inoculated into apple juice at a rate of 0.3 g / 100 g juice, and fermented at 30° C. for 48 h.

[0037] Preparation of plant lactobacillus inoculum: streak a plant lactobacillus (CICC 21801) bacterial solution preserved in 50% glycerol at -80°C on an MRS agar medium, incubate at 37°C for 24 hours to obtain a single colony, pick a single colony with an inoculating loop and place it in an MRS broth medium, incubate at 37°C for 24 hours to obtain the bacterial solution, centrifuge and collect the bacterial bodies, wash twice with sterile water, and freeze-dry in a freeze dryer to obtain freeze-dried bacterial powder.

[0038] Example 3

[0039] Other conditions were the same as those in Examples 1 and 2. The Kluyveromyces marxianus and Lactobacillus plantarum powders were mixed evenly in a ratio of 1:1, 0.3 g / 100 g juice was inoculated into apple juice, and fermented at 30° C. for 48 h.

[0040] Example 4

[0041] The other conditions were the same as those in Examples 1 and 2. The Kluyveromyces marxianus and Lactobacillus plantarum powders were mixed evenly in a ratio of 1:2, inoculated into apple juice at a rate of 0.3 g / 100 g juice, and fermented at 30° C. for 48 h.

[0042] Comparative Example 1

[0043] Other conditions were the same as those in Examples 1 and 2. The Lactobacillus plantarum powder was inoculated into apple juice at a ratio of 0.3 g / 100 g juice and fermented at 30° C. for 48 h.

[0044] The physical and chemical indicators of fermented apple juice were determined, including pH, soluble solids, reducing sugars, total phenols, total flavonoids, and organic acids. The color and antioxidant activity of the fermented apple juice were determined, and sensory evaluation was performed. Lactobacillus plantarum CICC21801 was inoculated alone as the control group.

[0045] ① Determination of physical and chemical indicators: pH was determined using a pH meter. The fermented juice was taken at 0, 24, and 48 hours of fermentation and inoculated with Lactobacillus plantarum alone as the control group. The pH changes were as follows: Figure 2 As shown. The TSS value was determined by a saccharimeter, as shown in Table 1. After the above indicators were determined, the fermentation liquid was centrifuged (12,000r×2min) and the supernatant was stored in a -20℃ refrigerator for later use. The total phenol content in the fermented juice was determined by the Folin phenol method, the total flavonoid content in the fermented juice was determined by spectrophotometry, the total reducing sugar content was determined by the DNS method, and the organic acid content was determined by high performance liquid chromatography, as shown in Table 1.

[0046] ②Determination of antioxidant activity of fermented apple juice

[0047] Determination of DPPH free radical scavenging rate

[0048] Weigh 39.4 mg of DPPH, dissolve it in 95% ethanol and dilute it to a 10L volumetric flask to obtain a 1 mol / mL DPPH solution, and store it at -4°C away from light. Take 2 mL of the sample solution and mix it with 0.5 mL of DPPH solution and 0.5 mL of anhydrous methanol, respectively, and place it at room temperature away from light for 3 minutes, and measure its absorbance A at 517 nm. i and A j; Mix 2 mL of anhydrous methanol with 0.5 mL of DPPH solution, place at room temperature and away from light for 3 min, and measure the absorbance A at 517 nm using an enzyme marker c , all with anhydrous methanol as blank. DPPH free radical scavenging rate was calculated according to the formula.

[0049]

[0050] Determination of ABTS free radical scavenging rate

[0051] First, prepare 7mmol / LABTS working solution, accurately weigh 0.003g ABTS, dilute to 0.8mL with distilled water, weigh 0.001g potassium persulfate and dissolve it in 1.5mL distilled water to make 2mol / L potassium persulfate aqueous solution, then mix it to 1.6mL at a ratio of 1 (0.8mL): 1 (0.8mL) and place it at room temperature away from light for 12-16h, dilute with distilled water and adjust the absorbance to 0.700±0.005 for use. Add 200μL sample to 800μLABTS working solution, let it stand for 6min, and measure the absorbance value at 734nm with an enzyme marker. The results are expressed as Trolox equivalents (μmolTE / 100g juice) per 100g juice.

[0052] The ABTS free radical scavenging rate was calculated according to the formula.

[0053]

[0054] Where: A0—absorbance of distilled water and ABTS working solution at 734nm; A j —Absorbance of fermentation broth and ABTS working solution at 734 nm

[0055] ③ Sensory evaluation method: 13 teachers and student team members with relevant experience and background knowledge evaluated the color, aroma and taste of fermented apple juice. The specific evaluation criteria are shown in Table 3. The average score for each aspect was taken. The specific sensory evaluation results are shown in Table 4.

[0056] Experimental results:

[0057] 1. As shown in Table 1, the physicochemical indicators of probiotic fermented apple juice are shown in Table 1. The apple juice fermented by Kluyveromyces marxii and the apple juice fermented by the mixed inoculum of Kluyveromyces marxii and Lactobacillus plantarum had moderate acidity, and the contents of total phenols and total flavonoids were higher than those of unfermented apple juice and Lactobacillus plantarum fermented apple juice.

[0058] Table 1 Physical and chemical indicators of fermented apple juice

[0059]

[0060] 2. Antioxidant activity of probiotic fermented apple juice Figure 3 , 4 As shown in the figure. The DPPH free radical scavenging ability and ABTS free radical scavenging rate of apple juice fermented by Kluyveromyces marxensis increased by 1.18 times and 1.35 times compared with the original apple juice, and increased by 1.12 times and 1.12 times compared with the apple juice fermented by Lactobacillus plantarum alone; the DPPH free radical scavenging ability and ABTS free radical scavenging rate of apple juice fermented by Kluyveromyces marxensis and Lactobacillus plantarum powder in a ratio of 2:1 increased by 1.19 times and 1.38 times compared with the original apple juice, and increased by 1.12 times and 1.15 times compared with the apple juice fermented by Lactobacillus plantarum alone. It may be that the mixed fermentation produced a higher content of phenolic compounds, and phenolic compounds have been proven to be good reductants and free radical scavengers, mainly related to their hydrogen atom transfer or electron transfer to free radicals. Fermentation of Kluyveromyces marxensis alone or mixed fermentation of Kluyveromyces marxensis and Lactobacillus plantarum can significantly improve the antioxidant activity of apple juice.

[0061] 3. The sensory evaluation of probiotic fermented apple juice is shown in Table 3. The apple juice fermented by Kluyveromyces marxii and Lactobacillus plantarum has improved aroma, color, tissue morphology and taste compared with the original apple juice, and has a high public acceptance, ranking first among these embodiments. The public acceptance of apple juice fermented by Kluyveromyces marxii alone is also good, ranking second among these embodiments.

[0062] Table 2 Sensory evaluation standards for fermented apple juice

[0063]

[0064] Table 3 Sensory evaluation results of fermented apple juice in different implementation modes

[0065]

[0066]

[0067] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing probiotic fermented apple juice, characterized in that: The steps include: (1) Apple juice; (2) Enzymatic hydrolysis; (3) Sterilization; (4) preparing bacterial agents; (5) Inoculation and fermentation: Activated Kluyveromyces marxii CICC 32017, or Kluyveromyces marxii CICC 32017 and Lactobacillus plantarum CICC 21801 are inoculated into the apple juice prepared in step (3) for fermentation.

2. The method for preparing probiotic fermented apple juice according to claim 1, characterized in that: The enzymatic hydrolysis method in step (2) is to add pectinase and cellulase in a mass ratio of 1-3:1-3, and the total amount of enzyme added is 0.2-0.5% of the mass of the apple juice. The enzymatic hydrolysis is carried out at 45-50° C. for 3-5 hours, and the apple juice is filtered to obtain the apple juice.

3. The method for preparing probiotic fermented apple juice according to claim 1, characterized in that: The sterilization temperature in step (3) is 80-100°C and the time is 10-20 minutes.

4. The method for preparing probiotic fermented apple juice according to claim 1, characterized in that: The preparation methods of the Kluyveromyces marxkei and Lactobacillus plantarum inoculants described in step (4) are respectively as follows: streaking a Kluyveromyces marxkei bacterial solution preserved with 50% glycerol at -80°C on a YPD agar medium, standing and culturing at 25-30°C for 48-72h to obtain a single colony, picking a single colony with an inoculating loop in a YPD liquid medium, standing and culturing at 25-30°C for 48-72h to obtain a propagated bacterial solution, centrifuging the bacterial solution to collect bacterial bodies, washing them twice with sterile water, and freeze-drying them in a freeze dryer to obtain freeze-dried bacterial powder; streaking a Lactobacillus plantarum bacterial solution preserved with 50% glycerol at -80°C on an MRS agar medium, standing and culturing at 30-37°C for 24-48h to obtain a single colony, picking a single colony with an inoculating loop in a MRS broth medium, standing and culturing at 30-37°C for 24-48h to obtain a single colony, centrifuging the bacterial solution to collect bacterial bodies, washing them twice with sterile water, and freeze-drying them in a freeze dryer to obtain freeze-dried bacterial powder.

5. The method for preparing probiotic fermented apple juice according to claim 1, characterized in that: The inoculation amount in step (5) is 0.1-0.4 g bacterial powder / 100 g juice.

6. The method for preparing probiotic fermented apple juice according to claim 1, characterized in that: When Kluyveromyces marxianus CICC 32017 and Lactobacillus plantarum CICC 21801 are mixed for use, the mass ratio of Kluyveromyces marxianus to Lactobacillus plantarum in the mixed strain fermentation is 1-2:1-2.

7. The method for preparing probiotic fermented apple juice according to claim 1, characterized in that: The fermentation conditions in step (5) are culturing in a constant temperature incubator at 25 to 30° C. for 48 to 72 hours.

8. Probiotic fermented apple juice obtained by the method for preparing probiotic fermented apple juice according to any one of claims 1 to 7.

9. Use of the probiotic fermented apple juice obtained by the method for preparing probiotic fermented apple juice according to any one of claims 1 to 7 in preparing antioxidant products.

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