A method for fermenting a flavored tangerine whole juice beverage using Lactobacillus plantarum

Abstract

The invention belongs to the application field of microorganisms or enzymes; in particular, it relates to a method for fermenting a flavored tangerine whole juice beverage by using plant lactobacillus, comprising the following steps: whole-fruit juicing of fresh tangerines, then adjusting the pH to 4.7-5.3, then pasteurizing, and then naturally cooling to room temperature; immediately inoculating the obtained juice naturally cooled to room temperature with lactic acid bacteria and then fermenting, ensuring that the initial concentration of the lactic acid bacteria after the juice is inoculated is 10 7 ~10 8 CFU / mL; the fermented tangerine juice is centrifuged, and the clarified liquid obtained by centrifugation is a flavor-enhanced fermented tangerine whole juice beverage. The method of the present invention can enhance the flavor of the tangerine whole juice beverage.

Description

Technical Field

[0001] The invention belongs to the application field of microorganisms or enzymes, and specifically relates to a method for fermenting a flavored tangerine whole juice beverage using Lactobacillus plantarum. Background Art

[0002] Mandarin orange is a plant of the genus Citrus in the Rutaceae family. It is one of the citrus fruits. Other similar fruits include orange, tangerine, grapefruit, lemon and other varieties. As one of the origins of citrus, my country has a wide citrus planting area and high yield. Citrus fruits are rich in nutrients such as amino acids, vitamins, minerals, and contain bioactive substances such as phenols, flavonoids, carotene, and coumarins, and are deeply loved by people. However, a large amount of peel residue is often produced during the processing of citrus juice products, accounting for about 40-60% of the total fruit volume, mainly including peel, part of the pulp, and a small amount of endocarp and residual membrane of vesicles. Studies have shown that citrus peel residue also contains rich nutrients and functional ingredients, such as pectin, flavonoids and cellulose. In recent years, with the continuous enhancement of people's health awareness, fruits with biological activity have received more and more attention from people. Better use of various parts of citrus fruits to produce products with higher nutritional value is a major trend in citrus juice processing.

[0003] However, traditional tangerine juice often has problems such as single flavor, easy oxidation and deterioration, and difficulty in maintaining fresh flavor for a long time during the production process. In recent years, more and more companies have begun to explore the use of fermentation technology to improve the preparation process of fruit juice beverages. Fermentation technology can not only reduce the undesirable flavor components (such as bitterness and astringency) in juice through the metabolism of microorganisms, but also produce rich aroma substances (such as esters, alcohols and acids). These aroma substances can significantly enhance the flavor level of juice and give it a unique aroma enhancement effect.

[0004] At present, some patents on fermented citrus juice beverages have been published, such as the invention of CN 114868855 "A method for preparing fermented citrus peel beverage from whole citrus peel", the invention of CN 117121998 "A citrus probiotic beverage and its preparation method", and the invention of CN 108308481 "A preparation process of probiotic fermented citrus beverage"; these patents mainly focus on controlling fermentation conditions, improving juice stability and sensory properties, etc. However, for the specific raw material of mandarin orange, especially the preparation method of flavored fermented mandarin orange beverage in the form of whole juice, there is still room for further research and improvement. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for improving the flavor of citrus whole juice by using fresh tangerine whole juice as a raw material and fermenting it by inoculating a preferred flavor-enhancing plant lactobacillus.

[0006] In order to solve the above technical problems, the present invention provides a method for fermenting a flavored tangerine whole juice beverage using Lactobacillus plantarum, comprising the following steps:

[0007] S1. Juice extraction:

[0008] The washed fresh tangerines are naturally dried (until there is no obvious water stain on the surface) and then the whole fruit is squeezed for juice. The collected juice is filtered (filtered) for clarification to obtain clarified juice;

[0009] Description: Whole fruit juice is to squeeze the peel and pulp of the tangerine together to obtain mixed juice;

[0010] S2. Adjust pH:

[0011] Adjusting the pH of the clarified juice obtained in step S1 to 4.7-5.3 to obtain pH-adjusted juice;

[0012] S3, activating and preparing lactic acid bacteria;

[0013] S4. Pasteurization:

[0014] The pH-adjusted juice obtained in step S2 is pasteurized (thermal sterilization at 94-96° C. for 9-11 min), and then naturally cooled to room temperature;

[0015] S5. Inoculation and fermentation:

[0016] The juice naturally cooled to room temperature obtained in step S4 is immediately inoculated with lactic acid bacteria for fermentation, and the initial concentration of lactic acid bacteria after the juice is inoculated is ensured to be 10 7 ~10 8 CFU / mL, the fermentation environment temperature was controlled at 28±0.5℃, and the fermentation cycle was controlled at 72±1h;

[0017] S6. Clarification of fermented juice:

[0018] The fermented tangerine juice obtained in step S5 is centrifuged at 7500-8500 rpm and 3-5° C. for 8-12 min (preferably 8000 rpm for 10 min). The clarified liquid obtained by centrifugation is a flavored fermented tangerine whole juice beverage.

[0019] The flavor-enhanced fermented tangerine whole juice beverage can be stored at -20°C.

[0020] As an improvement of the method of using plant lactobacillus to ferment and enhance the flavor of tangerine whole juice beverage of the present invention: the lactic acid bacteria is plant lactobacillus ( Lactobacillus plantarum )ZFM715.

[0021] Description: Lactobacillus plantarum ZFM715 was first activated at 37±0.5℃, and then lactic acid bacteria were prepared;

[0022] The utensils used for activating strains should be disposable or sterilized at high temperature first.

[0023] As a further improvement of the method for fermenting a flavored tangerine whole juice beverage using Lactobacillus plantarum of the present invention: a 40-mesh filter is used for filtration in step S1.

[0024] As a further improvement of the method for fermenting a flavored tangerine whole juice beverage using Lactobacillus plantarum of the present invention: in step S2, edible alkali (edible baking soda) and food-grade citric acid are used to adjust the pH.

[0025] As a further improvement of the method for producing a flavored tangerine whole juice beverage by fermenting with Lactobacillus plantarum of the present invention: the pH in step S2 is 4.8-5.0.

[0026] The present invention has the following technical advantages:

[0027] 1. The strain selected in the present invention is Lactobacillus plantarum (flavor-enhancing probiotic ZFM715) obtained after screening and comparison, which can effectively reduce the undesirable flavor substances in tangerine juice, while enhancing the rich aroma intensity of tangerine juice such as floral fragrance, sweet fragrance, and citrus fragrance, thereby improving the sensory flavor of tangerine juice.

[0028] 2. The raw material selected by the present invention is the whole tangerine fruit, including the peel and the pulp, which makes better use of various parts of the citrus fruit, retains the rich nutrients and bioactive substances in the peel, and meets the consumer's consumption concept of healthy food.

[0029] 3. The present invention uses the flavor-enhancing probiotic ZFM715 to ferment whole tangerine juice, which has low cost, short fermentation cycle and simple operation.

[0030] 4. The present invention uses GC-MS analysis method to monitor the flavor substance content of the flavor-enhanced fermented mandarin orange whole juice beverage, which is more scientific and objective than the results of human sensory analysis alone.

[0031] 5. The present invention is a method for improving the flavor of tangerine whole juice by using fresh tangerine whole juice as raw material and adopting flavor-enhancing plant lactobacillus fermentation technology.

[0032] In summary, the present invention provides a flavor-enhancing Lactobacillus plantarum fermentation technology, which is specifically used to improve the flavor quality of whole tangerine juice. The core is to accurately inoculate Lactobacillus plantarum into whole tangerine juice and control the appropriate temperature for fermentation, effectively adjust the composition of volatile compounds in the juice, significantly enhance and enrich the aroma of the juice, and achieve the purpose of flavor enhancement. The method of the present invention can effectively reduce the undesirable flavor components in the juice and produce rich aroma substances, which can be applied to the flavor improvement of whole tangerine juice and similar fruit and vegetable juices. In addition, the present invention also shows good universality and can be flexibly applied to the flavor optimization of a variety of fruit juices, which not only shortens the production cycle, but also ensures the effective control of costs and the simplicity and speed of the operation process. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0034] Selecting autochthonous lactic acid bacteria for co-inoculation in Chinese bayberry wine production:Stress response, starter culturesapplication and volatilomic study(Food Research international 178 (2024)11396)informed Lactobacillus plantarum ZFM715 (Lp. plantarum ZFM715), which was screened in bayberry juice.

[0035] The invention adopts fresh tangerines picked within 30 days.

[0036] Embodiment 1, a method for fermenting a flavored tangerine whole juice beverage using Lactobacillus plantarum, sequentially performing the following steps:

[0037] S1. Juice extraction:

[0038] Fresh, non-rotten, smooth-skinned tangerines without pests and mechanical damage are stalked, washed and naturally dried (until there is no obvious water stain on the surface), and then the whole fruit is squeezed, and the squeezed juice is filtered through a 40-mesh filter to clarify the juice, thereby obtaining clarified juice.

[0039] Description: Whole fruit squeezing is to squeeze the peel and pulp of tangerine together to obtain mixed juice; large particles of impurities and suspended matter can be removed through a 40-mesh filter to obtain clear and transparent tangerine juice, which is named clarified juice.

[0040] S2. Adjust pH:

[0041] The pH of the clarified juice obtained in step S1 is adjusted to 5.0 using edible alkali (NaHCO3) or food-grade citric acid to obtain a pH-adjusted juice.

[0042] S3. Preparation of lactic acid bacteria cells:

[0043] The utensils used to activate the strain need to be sterilized at high temperature (121℃ for 15 minutes);

[0044] Lactobacillus plantarum ZFM715 was inoculated on MRS solid culture medium and cultured at 37°C for 48 h to achieve activation of Lactobacillus plantarum ZFM715; then the activated single colony was picked out and inoculated into MRS broth, cultured at 37°C for 24 h and then subcultured, and the culture solution was inoculated into new MRS broth at an inoculation rate of 1% (volume %) to obtain a second-generation lactic acid bacteria suspension.

[0045] In order to ensure the effect of lactic acid bacteria, the second generation lactic acid bacteria suspension generally needs to ensure that the number of live bacteria is ≥10 9 CFU / mL; if the viable count of the second-generation lactic acid bacteria suspension cannot meet the above requirements, the second-generation lactic acid bacteria suspension is inoculated on MRS solid medium and cultured at 37°C for 48 h (i.e., the activation of Lactobacillus plantarum ZFM715 is repeated); and then the second-generation lactic acid bacteria suspension is prepared according to the above method; that is, the above activation and the preparation of the second-generation lactic acid bacteria suspension are repeated until the viable count of the obtained second-generation lactic acid bacteria suspension is ≥10 9 CFU / mL.

[0046] Note: The colony counting method can adopt the conventional blood cell counting method or the plate method. The plate method is to dilute with physiological saline and then spread and count.

[0047] The second generation lactic acid bacteria suspension (viable bacteria count ≥ 10 9 CFU / mL) was centrifuged at 6000 rpm at 4°C for 5 min, the supernatant was removed, and the bacteria were washed twice with physiological saline (0.9%). The washed bacteria were used for subsequent inoculation and fermentation.

[0048] S4. Pasteurization:

[0049] The pH-adjusted juice obtained in step S2 is pasteurized (thermal sterilization at 95° C. for 10 min), and then naturally cooled to room temperature.

[0050] S5. Inoculation and fermentation:

[0051] The juice obtained in step S4 and cooled naturally to room temperature is immediately inoculated with the lactic acid bacteria obtained in step S3, and the initial concentration of lactic acid bacteria in the juice after inoculation is ensured to be 10 7CFU / mL, the fermentation environment temperature was controlled at 28°C, and the fermentation cycle was controlled at 72h;

[0052] After the above-set fermentation time is reached, the pH value is about 4.25 and the sugar content is about 15.4; therefore, the fermentation is ended.

[0053] S6. Clarification of fermented juice:

[0054] The fermented tangerine juice obtained in step S5 is centrifuged at 8000 rpm for 10 min at low temperature (the centrifugation temperature is 4° C.), and the clarified liquid obtained by centrifugation is a flavor-enhanced fermented tangerine whole juice beverage.

[0055] Subsequently, aseptic filling is carried out immediately, and the product is immediately sealed and stored at -20°C to maintain the best flavor and quality of the product.

[0056] Description: Low-temperature centrifugation can remove bacteria and macromolecular precipitates in the fermentation broth, retaining the clear fermented tangerine juice with a strong aroma.

[0057] Embodiment 2: Compared with Embodiment 1, the following changes are made:

[0058] In step S5, the fermentation environment temperature is changed to 30°C and the fermentation time is changed to 60 hours. At this time, the pH value is about 3.93 and the sugar content is about 15.1; therefore, the fermentation is terminated.

[0059] Embodiment 3: Compared with Embodiment 1, the following changes are made:

[0060] The fermentation cycle was changed from 72h to 12h, 24h, 36h, 48h, 60h and 84h respectively.

[0061] The fermentation environment temperature remained unchanged at 28°C;

[0062] The rest is the same as Example 1.

[0063] The pH and sugar content values ​​corresponding to each fermentation time are shown in Table 1 below.

[0064] Table 1

[0065]

[0066] Experiment 1, Example 1 The clarified juice obtained in step S1 was subjected to GC / MS volatile component analysis:

[0067] 10 mL of sample was mixed with 3.0 g NaCl and 10 mL 4-methyl-2-pentanol (concentration 8.11 mg / mL) and incubated in a constant temperature water bath at 50°C for 30 min. After equilibrium, the SPME fiber coated with divinylbenzene / carboxymethyl / polydimethylsiloxane (DVB / CAR / PDMS) was inserted into the headspace bottle and extracted at 50°C for 30 min. The extraction head was then removed and inserted into the gas chromatograph injection port for GC-MS analysis.

[0068] The chromatographic parameters are as follows: the chromatographic column is a DB-WAX column (0.5 μm, 60 m × 0.25 mm, Agilent), the carrier gas is He (purity ≥99.999%), and the flow rate is 1.0 mL / min. The temperature was programmed to start at 35°C, hold for 3 min, increase to 160°C at a rate of 2°C / min, increase to 200°C at a rate of 3°C / min, increase to 240°C at a rate of 4°C / min and maintain for 5 min, and the injection mode was splitless. The instrument used was Agilent 7890A-5975C, Santa Clara, CA. The results obtained---the names and contents of flavor components (aroma substances) are described in Table 2 below.

[0069] Table 2

[0070]

[0071] Experiment 2: The flavor-enhanced fermented tangerine whole juice beverage obtained in Example 1 was subjected to GC / MS detection according to the method described in Experiment 1. The results obtained - the names and contents of flavor components (aroma substances) are shown in Table 3 below.

[0072] Table 3

[0073]

[0074] Experiment 3: The flavor-enhanced fermented mandarin orange whole juice beverage obtained in Example 2 was subjected to GC / MS detection according to the method described in Experiment 1. The names and contents of the obtained flavor components (aroma substances) are described in Table 4 below.

[0075] Table 4

[0076]

[0077] Note: The aroma descriptions in the above table were all found on the websites Flavornet and vcf-online (*).

[0078] Note: The components "γ-terpinene, terpinene, decanal and styrene" in Table 2 are prone to produce odor at high concentrations, which is an unpleasant flavor. Moreover, as common sense, dimethyl sulfide is a typical odor substance in pasteurized juice. In Tables 3 and 4, the contents of γ-terpinene, terpinene, decanal, styrene and dimethyl sulfide are significantly reduced or not detected, which shows that the technical solution of the present invention effectively reduces the unpleasant flavor in juice.

[0079] The (Z)-β-ocimene, eugenol, linalool and vinyl acetate components in Tables 3 and 4 are aroma substances. As common sense, they can produce citrus, floral, sweet and fruity odors, respectively. However, these components do not exist in Table 2 (or the content of these components in Table 2 is low). Therefore, it is shown that the technical solution of the present invention can produce rich aroma substances.

[0080] In addition, according to the comparison between Table 3 and Table 4, it can be seen that there are 95 aroma substances in Example 1, while there are only 79 aroma substances in Example 2, which shows that the aroma substances produced by the fermentation method of Example 1 are more abundant, and the concentration of substances with good flavor (floral fragrance, citrus fragrance, etc.) such as linalool, hexanol, eugenol, geraniol and (Z)-β-ocimene in Example 1 is significantly higher than that in Example 2. Relatively speaking, Example 1 is a preferred case.

[0081] Experiment 4: The flavor-enhanced fermented mandarin orange whole juice beverage obtained at different fermentation times in Example 3 was subjected to GC / MS detection according to the method described in Experiment 1. The names and contents of some of the flavor components (aroma substances) obtained were compared with those in Example 1 as shown in Table 5 below.

[0082] Table 5

[0083]

[0084] Comparative Experiment 1. The present invention also conducted the following comparative experiments during the invention process:

[0085] The strain in the above Example 1 was changed from Lp. plantarum ZFM715 to the following plant lactobacillus: CNCM I-2845, plant lactobacillus B7, plant lactobacillus Lactobacillus plantarum (CCTCC NO: M 20232682), plant lactobacillus Lp goji (CGMCC NO.16221), and the rest was the same as in Example 1. The results obtained were: the linalool content in the flavor-enhanced fermented tangerine whole juice beverage obtained after fermentation by the above strains was not significantly different from the linalool content in Table 2, while the content of decanal increased relative to Table 2, especially the decanal content in the tangerine juice fermented by Lactobacillus plantarum B7 was as high as about 530 μg / L, and the styrene content was also basically close to the content described in Table 2. Therefore, it is shown that only the plant lactobacillus ZFM715 of the present invention can effectively reduce the undesirable flavor substances in tangerine juice, while enhancing the rich aroma intensity of the tangerine juice such as floral fragrance, sweet fragrance, citrus fragrance, etc., thereby improving the sensory flavor of the tangerine juice.

[0086] Comparative Example 1: Step S2 of Example 1 is cancelled, that is, Step S4 is changed to: pasteurizing the clarified juice obtained in Step S1, and the rest is the same as Example 1.

[0087] Comparative experiment 2

[0088] After the inoculation of step S5 of Example 1 is completed (the initial concentration of lactic acid bacteria in the juice after inoculation is 10 7 CFU / mL) for 72 h, according to the conventional OD 600 The lactic acid bacteria content was detected by the method (monitoring every 12 h), and the results were 0.170 (12 h), 0.184 (24 h), 0.221 (36 h), 0.782 (48 h), 0.762 (60 h), and 1.039 (72 h).

[0089] After the inoculation of step S5 of comparative example 1 was completed (the initial concentration of lactic acid bacteria in the juice after inoculation was 10 7 CFU / mL) for 72 hours, according to the conventional OD 600 The lactic acid bacteria content was detected by the method, and the results were 0.181 (12 h), 0.159 (24 h), 0.151 (36 h), 0.155 (48 h), 0.123 (60 h), and 0.261 (72 h).

[0090] Therefore, it can be seen that the technical solution of Example 1 is more suitable for the growth of lactic acid bacteria.

[0091] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for a flavored tangerine whole juice beverage using Lactobacillus plantarum fermentation, characterized in that: The method uses whole tangerine fruit as raw material, wherein the whole tangerine fruit is the peel and pulp of the tangerine; and comprises the following steps: S1. Juice extraction: The washed fresh tangerines are naturally dried and then the whole fruit is squeezed for juice, and the collected juice is filtered for clarification to obtain clarified juice; S2. Adjust pH: Adjusting the pH of the clarified juice obtained in step S1 to 4.8-5.0 to obtain pH-adjusted juice; S3. Activation to obtain lactic acid bacteria: Lactobacillus plantarum ( ‌Lactobacillus plantarum )ZFM715; S4. Pasteurization: The pH-adjusted juice obtained in step S2 is pasteurized and then naturally cooled to room temperature; S5. Inoculation and fermentation: The juice naturally cooled to room temperature obtained in step S4 is immediately inoculated with lactic acid bacteria for fermentation, and the initial concentration of lactic acid bacteria after the juice is inoculated is ensured to be 10 7 ~10 8 CFU / mL, the fermentation environment temperature was controlled at 28±0.5℃, and the fermentation cycle was controlled at 72±1h; S6. Clarification of fermented juice: The fermented tangerine juice obtained in step S5 is centrifuged at 7500-8500 rpm and 3-5° C. for 8-12 min, and the clarified liquid obtained by centrifugation is a flavor-enhanced fermented tangerine whole juice beverage.

2. The method for producing a flavored tangerine whole juice beverage by fermenting with Lactobacillus plantarum according to claim 1, characterized in that: The filtration in step S1 is performed using a 40-mesh filter.

3. A method for producing a flavored tangerine whole juice beverage by fermenting with Lactobacillus plantarum according to claim 2, characterized in that: In step S2, edible alkali and food-grade citric acid are used to adjust the pH.

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