A pure oat-based fermented beverage and a method for preparing the same

Abstract

The application discloses a pure oat-based fermented beverage and a preparation method thereof, and belongs to the technical fields of microorganisms and fermentation. The pure oat beverage is used as a fermentation raw material, and lactococcus lactis is added to perform fermentation, so that the content of beta-glucan in the oat beverage is increased by 40% to 70%, and the taste of the oat beverage is improved. The ratio of sweet amino acids to bitter amino acids is (1-1.2):1. Compared with other similar products obtained by fermentation of pure plant bases, the product is natural and delicious, has a long survival time in storage, does not need complex processes and expensive equipment, has low product manufacturing cost, and has good practical value and application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a pure oat-based fermented beverage and a preparation method thereof, and belongs to the fields of microbiological technology and fermentation technology. BACKGROUND

[0002] Oats are cultivated and consumed all over the world and are a rich source of energy, vitamins and minerals, and have high nutritional value, containing various nutrients such as proteins, fats, phenolic compounds, minerals and beta-glucans, which play an important role in preventing cardiovascular diseases, colon cancer, type II diabetes and many other diseases. Oats contain 18 kinds of amino acids including essential amino acids for human body, among which the contents of glutamic acid, phenylalanine, lysine and leucine rank first among oats, wheat, buckwheat, barley and rice, the content of lysine is 2 times higher than that of rice and wheat, and compared with other types of grains, the amino acid ratio of oats is reasonable and balanced.

[0003] Meanwhile, oats are rich in dietary fiber, the main component of which is beta-glucan, which has the effects of lowering cholesterol and preventing cardiovascular diseases, and another important function of beta-glucan is its prebiotic function in the intestinal mucosa, which supports the growth of beneficial microflora and produces short-chain fatty acids such as propionic acid, butyric acid and acetate, which have multiple effects such as supporting host health, regulating lipid metabolism disorders and reducing oxygen radical generation. Beta-glucan is a non-starch polysaccharide composed of D-glucose connected by beta-glucoside bonds, which exists widely in nature, and its main sources are cereals, bacteria and fungi. Based on the various benefits of beta-glucan to human health, more and more products containing beta-glucan active ingredients are being developed and commercially produced. The main methods for producing beta-glucan are chemical synthesis, enzyme catalysis and biosynthesis. Among them, biosynthesis is mainly through microbial metabolism to synthesize beta-glucan, and fermentation is a commonly used method in biosynthesis, which has the advantages of small pollution, low cost, mild reaction conditions and high product yield, and has very broad application prospects.

[0004] At present, oats are mainly used as fermentation substrate in combination with milk, and there are problems such as odor, contamination by miscellaneous bacteria and short shelf life in products fermented with pure oats. The taste of oat beverage is related to the content of flavor amino acids in it, sweet amino acids are positively correlated with the sensory quality of oat beverage, and bitter amino acids are negatively correlated with the sensory quality of oat beverage, and the content of sweet amino acids and bitter amino acids in existing pure oat-based beverages is unbalanced, resulting in poor taste of pure oat-based beverages.

[0005] Therefore, it is urgent to develop a pure oat-based fermented beverage with a proper ratio of sweet amino acids to bitter amino acids and good taste, so as to improve the health value of the fermented oat beverage on the basis of meeting the flavor. SUMMARY

[0006] The application provides application of Lactococcus lactis in preparation of an oat fermented beverage.

[0007] The application provides application of Lactococcus lactis in preparation of an oat fermented beverage.

[0008] In one embodiment, the Lactococcus lactis is added in an amount of (1-100) × 10 6 CFU / ml of oat enzymatic hydrolysate.

[0009] In one embodiment, the oat enzymatic hydrolysate is prepared by one-stage enzymatic hydrolysis and two-stage enzymatic hydrolysis of oat; the one-stage enzymatic hydrolysis is carried out by using amylase, the amylase is mesophilic amylase BAN 480L and / or Fungamyl 800L amylase, and the amylase is added in an amount of 0.5‰-1.5‰ of the mass of oat; the two-stage enzymatic hydrolysis is carried out by using a composite enzyme, the composite enzyme comprises two or more of Fungamyl 800L amylase, flavor protease, glucoamylase and alkaline protease, and the composite enzyme is added in an amount of 0.5‰-4.5‰ of the mass of oat.

[0010] The application further provides a method for preparing a pure oat-based beverage, comprising the following steps:

[0011] (1) preparing oat enzymatic hydrolysate: selecting oat kernels, soaking the oat kernels in water at a ratio of 1:1-1:3 for 2-6 hours, adding 0.5‰-1.5‰ of amylase (calculated by the mass of oat), uniformly beating the oat kernels, then finely grinding the oat kernels in a colloid mill to obtain oat fine slurry, and subjecting the oat fine slurry to one-stage enzymatic hydrolysis at a temperature of 65-85 ℃ for 0.5-1.5 hours; heating to inactivate the enzyme; adding a composite enzyme to perform two-stage enzymatic hydrolysis at a temperature of 40-60 ℃ for 0.5-1.5 hours; heating to inactivate the enzyme; filtering the oat slurry after inactivation of the enzyme to remove residues, obtaining oat liquid; adjusting the solid content of the oat liquid to 15%-30%, homogenizing to obtain oat enzymatic hydrolysate, and sterilizing the oat enzymatic hydrolysate at 80-120 ℃, and cooling after sterilization for standby use.

[0012] (2) Preparation of fermentation inoculum: under sterile conditions, activate the glycerol tube of Lactococcus lactis preserved in the laboratory, inoculate them into MRS liquid medium, and cultivate at 30°C for 12-24 h. After successful activation, repeat the cultivation at 30°C for 12-24 h, and then pick the bacterial liquid with an inoculation loop. After 24-72 h of cultivation on a test tube slant prepared from MRS solid medium, pick a single colony of Lactococcus lactis obtained on the slant, cultivate in a liquid medium at 30°C for 12-24 h, repeat the operation for 2-3 times, and then take 1% of the liquid medium for expansion cultivation. Centrifuge the obtained bacterial liquid at 4°C at a speed of 5000 r / min for 10-15 min, discard the supernatant, and retain the bacterial slurry. Wash the bacterial slurry with sterile normal saline for 3 times, and finally resuspend it with an equal volume of normal saline to obtain a Lactococcus lactis bacterial suspension.

[0013] (3) Inoculation fermentation: add the Lactococcus lactis bacterial suspension to the sterilized oat enzymatic hydrolysate at an inoculation amount of (0.3-3) x 10 6 CFU / ml, shake well, and then place in a 25°C incubator for cultivation for 8-12 h. After fermentation, place in a 4°C refrigerator for post-ripening for 12-36 h to obtain a fermented oat beverage.

[0014] In an embodiment, the soaking in the aforementioned step (1) refers to normal-temperature soaking, the enzyme in the one-stage enzymolysis refers to mesophilic amylase BAN 480L and / or Fungamyl 800L amylase, the addition amount is 0.5‰-1.5‰, the complex enzyme in the two-stage enzymolysis includes two or several of Fungamyl 800L amylase, flavor protease, saccharifying enzyme, and alkaline protease, the addition ratio is 0.5‰-4.5‰, the heating and enzyme-killing condition is 90-100°C for 10-20 min, the filter screen aperture for filtering out the residue is 150-300 meshes, and the homogenization pressure is 25-45 Mpa.

[0015] In an embodiment, the bacterial concentration of the Lactococcus lactis fermentation inoculum obtained in the aforementioned step (2) is ≥1 x 10 6 CFU / mL.

[0016] In an embodiment, the Lactococcus lactis is one or several of Lactococcus lactis CICC 23610, Lactococcus lactis lactis subsp. CICC 20209, Lactococcus lactis cremoris subsp. CICC 20406, Lactococcus lactis CICC 20538, and Lactococcus lactis lactis subsp. CICC 21029.

[0017] In an embodiment, the amylase is mesophilic amylase BAN 480L and / or Fungamyl 800L amylase.

[0018] In an embodiment, the amount of the Lactococcus lactis added in the aforementioned step (3) is (0.3-3) x 10 6 CFU / ml of the oat enzymatic hydrolysate.

[0019] In an embodiment, the aforementioned selected Lactococcus lactis fermenting agent is screened from different commercial bacteria, and the oat beverage is fermented under respective suitable fermentation conditions, and the content of beta-glucan in the fermented oat beverage is determined after the post-ripening is completed, and the Lactococcus lactis CICC 23610 capable of increasing the content of beta-glucan by the largest amount is preferably obtained.

[0020] The application also provides a pure oat-based beverage prepared by the method, and the ratio of sweet amino acids to bitter amino acids in the beverage is (1-1.2):1.

[0021] Advantages:

[0022] (1) The application uses a pure oat beverage as a fermentation raw material, adds Lactococcus lactis for fermentation, and optimizes the fermentation agent to improve the taste of the oat beverage and increase the content of beta-glucan in the oat beverage, and the product obtained by fermentation of a pure plant base has natural and delicious taste.

[0023] (2) The application increases the content of free amino acids in the oat beverage by 15-20%, the ratio of sweet amino acids to bitter amino acids is (1-1.2):1, and the oat beverage has good taste; the content of beta-glucan in the oat beverage is effectively increased by 40%-70%, and the content of organic acid is increased by 20-30%.

[0024] (3) The number of viable Lactococcus lactis in the pure oat-based beverage obtained by the application is 9.87 log CFU / mL, which has a probiotic property.

[0025] (4) The method of the application is simple and convenient to operate, and the pure strain fermentation of Lactococcus lactis has no risk of contamination by miscellaneous bacteria, and the activity of beneficial bacteria is high, and there is no need for complex process and expensive equipment, the product manufacturing cost is low, and the application has good practical value and application prospect. DETAILED DESCRIPTION

[0026] The sweet amino acids involved in the application include serine, histidine, glycine, threonine, proline, and alanine.

[0027] The bitter amino acids involved in the application include arginine, valine, methionine, isoleucine, and leucine.

[0028] Example 1: Method for preparing a pure oat-based fermented beverage by using Lactococcus lactis

[0029] The embodiment is a method for improving the taste of oat beverage and increasing the content of beta glucan in oat beverage by using Lactococcus lactis fermentation, which specifically comprises the following steps:

[0030] (1) Select full and complete oat kernels, weigh, wash 3-5 times with tap water, add a certain amount of deionized water to meet the ratio of oat kernels: water = 1:1-1:3, soak for 3-6 hours at room temperature, then put into a wall breaking machine, add 1‰ Novozymes medium temperature amylase based on the mass of oats, evenly beat and pulp, then put the oat pulp into a colloid mill for 1-2 passes of rough grinding and 3-5 passes of fine grinding to achieve sufficient fineness.

[0031] (2) Add 1‰ Novozymes medium temperature amylase BAN480 (enzyme activity 480 KNU / g) based on the mass of oats to the oat fine pulp, and perform one-stage enzymolysis at 75℃ for 1h. After the enzymolysis is completed, boil to inactivate the enzyme for 15min. After inactivation, cool down, and then add 1‰ Novozymes amylase (enzyme activity 800 FAU / g), 1‰ flavor protease from Shaanxi Haoying (enzyme activity 600000 U / g), 1‰ Angel glucose isomerase (enzyme activity 500000 U / g), and 0.5‰ Youpuk alkaline protease (enzyme activity 200000 U / g) based on the mass of oats, and perform two-stage enzymolysis at 40-60℃ for 1h. After the enzymolysis is completed, boil to inactivate the enzyme for 15min.

[0032] (3) Filter the enzymolyzed oat pulp with a 150-300 mesh filter screen to remove residues and obtain oat liquid. Adjust the solid content to 15-30%, and then homogenize 2-3 times at a pressure of 30-40Mpa to obtain oat enzymolysis liquid. Sterilize the oat beverage at 100℃ for 15min, and then bottle and store in a cold storage for standby.

[0033] (4) After being activated for 2 generations at 30℃ in MRS liquid medium, a ring of Lactococcus lactis CICC 23610 is streaked on a MRS solid medium slope and cultured at 30℃ for 24-72h. The strain on the slope is streaked into MRS liquid medium and cultured at 30℃ for 24h. After repeating the operation for 2-3 times, 1% of the volume of the bacterial liquid is inoculated into a conical flask with a liquid volume of 49mL for expansion culture for 12-24h. The obtained bacterial liquid is centrifuged at 3000-5000r / min at 4℃ for 10-15min. The supernatant is discarded and the bacterial slurry is repeatedly washed with sterile normal saline for 3 times. Finally, the bacterial slurry is resuspended with an equal volume of normal saline to obtain a Lactococcus lactis bacterial suspension (strain concentration ≥1×10 7 CFU / mL).

[0034] (5) The Lactococcus lactis bacterial suspension is inoculated into the oat beverage at a concentration of (0.3-3)×10 6The inoculum of 1 x 10 CFU / ml was inoculated into the sterilized oat enzymatic hydrolysate, and then fermented at 25°C for 10 h. After fermentation, the fermented oat beverage was matured at 4°C for 24 h.

[0035] (6) The content of β-glucan in the fermented oat beverage after maturation was determined. The detection method of β-glucan content: the prepared oat beverage was directly detected by the β-glucan [mixed-linkage] detection kit of Megazyme, and the specific steps were as follows:

[0036] 3 mL of sample was taken, boiled in a water bath for 5 min, and then cooled to room temperature. Then 8-10 mL of 95% ethanol was added, and then centrifuged at 4000 rpm for 15 min. The supernatant was discarded, 8-10 mL of 50% ethanol was added, and then centrifuged at 4000 rpm for 15 min. The supernatant was discarded. 4 mL of sodium phosphate (20 mM, pH 6.5) and 0.2 mL of lichenase solution (50 U / mL) were added to the precipitate, and then incubated at 50°C for 1 h. Then, 5 mL of sodium acetate buffer (200 mM, pH 4.0) was added, and the test tube was equilibrated to room temperature. The supernatant (0.1 mL; 1000g, 10 min) after centrifugation was hydrolyzed with 0.1 mL of β-glucosidase solution, 3 ml of GOPOD reagent was added, and the absorbance of the resulting solution was measured at 510 nm after incubation at 50°C for 20 min.

[0037] The mechanism of the determination of the content of β-glucan is as follows: first, β-glucan is decomposed into β-oligoglucose by lichenase; second, β-oligoglucose is decomposed into D-glucose by β-glucosidase; third, D-glucose is converted into D-gluconate by glucose oxidase; and finally, D-gluconate is converted into a red quinone compound by peroxidase, p-hydroxybenzoic acid, and 4-aminoantipyrine.

[0038] Table 1 Sensory indicators of fermented oat beverage

[0039]

[0040] Example 2: Method for preparing pure oat-based fermented beverage using different Lactococcus lactis

[0041] The specific implementation method is referred to Example 1, and the difference is that different Lactococcus lactis strains are used to prepare pure oat-based fermented beverage, the content of β-glucan, free amino acids, organic acids and viable bacteria in the fermented beverage are detected, and sensory evaluation is performed, and the results are shown in Table 2 (in the previous test and sensory evaluation, the ratio of sweet amino acids to bitter amino acids is (1-1.2):1, and the flavor of the oat beverage is better).

[0042] Table 2 Oat beverage fermented by different Lactococcus lactis

[0043]

[0044] Example 3: Method for preparing pure oat-based fermented beverage with different enzyme species

[0045] The detailed procedure is according to Example 1, except that different enzyme species were used in the one-stage enzymatic hydrolysis to prepare the pure oat-based beverage, and the content of β-glucan, free amino acids, organic acids and viable bacteria in the fermented beverage were determined, and sensory evaluation was performed, and the results are shown in Table 3.

[0046] Table 3 Oat beverage prepared by different amylase hydrolysis

[0047]

[0048] Example 4: Method for preparing pure oat-based fermented beverage by changing the amount of amylase in one-stage enzymatic hydrolysis

[0049] The detailed procedure is according to Example 1, except that the amount of amylase was changed in the one-stage enzymatic hydrolysis to prepare the pure oat-based beverage, and the content of β-glucan, free amino acids, organic acids and viable bacteria in the fermented beverage were determined, and sensory evaluation was performed, and the results are shown in Table 4.

[0050] Table 4 Oat beverage prepared by adjusting the amount of amylase

[0051]

[0052] Example 5: Changing the enzymatic hydrolysis time

[0053] The detailed procedure is according to Example 1, except that the enzymatic hydrolysis time of amylase in step 2 one-stage enzymatic hydrolysis was changed, and the content of β-glucan, free amino acids and viable bacteria in the oat beverage were determined, and sensory evaluation was performed, and the results are shown in Table 9.

[0054] Table 5 Comparison of fermented oat beverages prepared by different enzymatic hydrolysis times

[0055]

[0056] Example 6: Changing the fermentation time

[0057] The detailed procedure is according to Example 1, except that the fermentation time in step 5 was changed, and the content of β-glucan, free amino acids and viable bacteria in the oat beverage were determined, and sensory evaluation was performed, and the results are shown in Table 8.

[0058] Table 6 Comparison of fermented oat beverages prepared by different fermentation times

[0059]

[0060] Comparative Example 1: No fermentation

[0061] The specific implementation is according to Example 1, except that steps (4) and (5) are omitted, and the content of β-glucan, free amino acids and organic acids in the oat beverage is detected. The results show that the content of β-glucan is 94% lower than that of Example 1, the content of free amino acids is 16.86% lower, the ratio of sweet-tasting amino acids to bitter-tasting amino acids is 1:1.3, and the content of organic acids is 25.85% lower.

[0062] Table 7 Comparison of the content of β-glucan in the fermented oat beverage prepared in Example 1 and Comparative Example 1

[0063]

[0064] Comparative Example 2: Change of strain

[0065] The specific implementation is according to Example 1, except that Lactobacillus bulgaricus CICC 20353, Streptococcus thermophilus CICC 20375, Lactobacillus plantarum CICC 22703, Lactobacillus casei CICC 20286, Lactobacillus helveticus CICC 20275 and Lactobacillus rhamnosus CICC 6001 are respectively used to replace Lactococcus lactis, and the content of β-glucan, free amino acids and viable bacteria in the oat beverage is detected, and sensory evaluation is performed. The results show that the content of β-glucan is 4-57% lower than that of Example 1, and the content of free amino acids is 8-14% lower. The oat beverage fermented by Lactococcus lactis has a higher viable bacteria count, indicating that the pure oat-based beverage can provide nutritional ingredients for the growth of Lactococcus lactis.

[0066] 20275 and Lactobacillus rhamnosus CICC 6001 are respectively used to replace Lactococcus lactis, and the content of β-glucan, free amino acids and viable bacteria in the oat beverage is detected, and sensory evaluation is performed. The results show that the content of β-glucan is 4-57% lower than that of Example 1, and the content of free amino acids is 8-14% lower. The oat beverage fermented by Lactococcus lactis has a higher viable bacteria count, indicating that the pure oat-based beverage can provide nutritional ingredients for the growth of Lactococcus lactis.

[0067] Table 8 Comparison of the fermented oat beverage prepared by different strains

[0068]

[0069] Comparative Example 3: Change of fermentation temperature

[0070] The specific implementation is according to Example 1, except that the fermentation temperature in step 5 is changed, and the content of β-glucan, free amino acids and viable bacteria in the oat beverage is detected, and sensory evaluation is performed, and the results are shown in Table 7.

[0071] Table 9 Comparison of the fermented oat beverage prepared by different fermentation temperatures

[0072]

[0073]

[0074] Although the present application has been disclosed in its preferred embodiments with reference to the accompanying drawings, it is not intended to limit the present application thereto, and various modifications and alterations can be made thereto by those skilled in the art without departing from the spirit and scope of the present application, and the scope of protection of the present application should be defined by the appended claims.

Claims

1. A process for the preparation of a pure oat based fermented beverage, characterized in that, The method comprises the following steps: (1) Beating: mix the oat kernels with water at a ratio of 1:1 to 1:3, and add 1 ‰ amylase based on the mass of the oat kernels, beat, and obtain oat fine pulp; (2) One-stage enzymolysis: add 1 ‰ amylase based on the mass of the oat kernels to the oat fine pulp obtained in step (1), and perform enzymolysis at 60-80 ℃ for 1 h to obtain enzymolysis liquid 1; (3) Two-stage enzymolysis: add 1 ‰ amylase, 1 ‰ flavor protease, 1 ‰ saccharifying enzyme, and 0.5 ‰ alkaline protease based on the mass of the oat kernels to the enzymolysis liquid 1 obtained in step (2), and perform enzymolysis at 40-60 ℃ for 1 h to obtain enzymolysis liquid 2; (4) Filtration: filter and homogenize the enzymolysis liquid 2 to obtain oat enzymolysis liquid; (5) Fermentation: inoculate Lactococcus lactis into the oat enzymolysis liquid in step (4), and perform fermentation at 20-30 ℃ for 10 h to obtain oat fermentation liquid; (6) Post-ripening: post-ripen the oat fermentation liquid in step (5) at 4 ℃ for 12-36 h; The inoculation amount of the Lactococcus lactis in step (5) is (0.3-3) x 10 6 CFU / ml oat enzymatic hydrolysate; The amylase in step (2) is a medium-temperature amylase; The pore size of the filtration in step (4) is 150-300 mesh; The Lactococcus lactis in step (5) is any one of Lactococcus lactis CICC 23610, Lactococcus lactis subsp. lactis CICC 20209, Lactococcus lactis subsp. cremoris CICC 20406, Lactococcus lactis CICC 20538, and Lactococcus lactis subsp. lactis CICC 21029.

2. A pure oat-based beverage prepared by the preparation method in claim 1.

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