A Lactobacillus starter culture and its application in the fermentation of guava juice

Abstract

The present invention provides a Lactobacillus starter and its application in the fermentation of guava juice. The Lactobacillus starter includes one or more of Lactiplantibacillus plantarum HNU082, Lactiplantibacillus plantarum HNU072, Lactiplantibacillus plantarum HNU503, and Lactobacillus mucosae HNU508. The present invention uses Lactobacillus to ferment guava juice, which has a positive effect on improving the flavor and taste of guava juice, extending the shelf life of guava juice, and enhancing the hypoglycemic functional characteristics of guava juice. It can further guide the fermentation and processing technology of guava juice and improve its economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation and relates to a lactobacillus fermentation agent and application thereof in guava juice fermentation. Background Art

[0002] Guava (also known as guava) is a nutritious tropical fruit rich in protein, dietary fiber, flavonoids, polyphenols, vitamins, minerals and other active ingredients. Studies have shown that these active ingredients have multiple physiological functions such as hypoglycemic, anti-oxidant, anti-inflammatory, antibacterial, anti-cancer, improving intestinal flora, delaying aging, maintaining cardiovascular health, nerve function and overall physiological balance. However, fresh guava juice is not resistant to storage and is easily contaminated by microorganisms and pathogens. At present, the shelf life of guava juice is mainly extended by heat treatment and non-heat treatment. Although the cost of heat treatment is low, it will destroy the nutrients in the juice and reduce the physiological activity of guava juice; while non-heat treatment can maximize the protection of nutrients and sensory properties, but it is expensive and cannot be mass-produced. Therefore, it is necessary to develop a low-cost processing technology that can maintain the nutrients of guava juice.

[0003] Lactobacillus fermentation has the advantages of safety, effectiveness and low cost, and has gradually become a new type of juice processing method. Lactobacillus fermentation can not only improve the nutritional value and functional activity of juice, but also prolong its storage life. Among them, the content of organic acids, sugars and phenolic substances in black cherry juice and sea buckthorn juice changed after lactobacillus fermentation, thereby improving the flavor of the juice, while after lactobacillus fermentation of kiwi juice, the content of polyphenols and flavonoids increased, and the antioxidant capacity increased accordingly. However, there are few studies on the fermentation effect of lactobacillus from different sources on juice, and there is also less attention on the study of metabolites of fermented guava juice. Summary of the invention

[0004] The present invention aims to provide a lactobacillus fermentation agent and application thereof in guava juice fermentation. The lactobacillus fermentation of guava juice has a positive effect on improving the flavor and taste of the guava juice, extending the shelf life of the guava juice, and enhancing the hypoglycemic functional characteristics of the guava juice, and can further guide the fermentation processing technology of the guava juice and improve its economic benefits.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] The present invention provides a lactobacillus fermentation agent, comprising one or more of Lactobacillus plantarum HNU082, Lactobacillus plantarum HNU072, Lactobacillus plantarum HNU503 and Lactobacillus mucilaginosus fermentation HNU508, wherein:

[0007] Lactobacillus plantarum HNU082 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on March 9, 2021, with the deposit number GDMCC NO: 61552, deposit address: 5th Floor, Laboratory Building, Provincial Institute of Microbiology, No. 100, Xianlie Middle Road, Guangzhou, China, taxonomic name: Lactiplantibacillus plantarum;

[0008] Lactobacillus plantarum HNU072 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on April 22, 2024, with the deposit number GDMCC NO: 64545, deposit address: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, taxonomic name: Lactiplantibacillus plantarum;

[0009] Lactobacillus plantarum HNU503 was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on April 22, 2024, with the deposit number GDMCC NO: 64547. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The taxonomic name is Lactiplantibacillus plantarum.

[0010] Fermented mucus Lactobacillus HNU508 was deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on April 22, 2024, with the deposit number GDMCC NO: 64548. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. The taxonomic name is Limosilactobacillus fermentum.

[0011] The present invention also provides a guava juice fermented beverage, which is fermented with the above-mentioned lactobacillus starter using guava juice as a fermentation substrate.

[0012] Preferably, the lactobacillus fermenter is resuspended in sterile physiological saline to a concentration of 10^8-10^9 CFU / mL, the inoculation amount in guava juice is 3%-10% V / V, and the fermentation conditions are: fermentation at 37°C for 24-48 hours; the guava juice is pasteurized guava juice.

[0013] More preferably, the inoculation amount of the Lactobacillus fermentation agent in the guava juice is 4% V / V, and the fermentation conditions are: fermentation at 37° C. for 36 hours.

[0014] The present invention further provides a method for preparing a fermented guava juice beverage, comprising: inoculating the above-mentioned lactobacillus ferment starter into pasteurized guava juice, and fermenting at 37 °C for 24 to 48 hours to prepare fermented guava juice.

[0015] Preferably, the lactobacillus ferment starter is resuspended in sterile physiological saline to a concentration of 10^8 - 10^9 CFU / mL, and then inoculated into pasteurized guava juice at an inoculation amount of 3% - 10% V / V.

[0016] The present invention also provides the application of the above-mentioned lactobacillus ferment starter in improving the flavor and function of fermented guava juice beverages.

[0017] Preferably, the application includes increasing the contents of total polyphenols and total flavonoids in the fermented guava juice beverage.

[0018] Preferably, the application includes increasing the antioxidant activity and α-Glu inhibitory activity of the fermented guava juice beverage.

[0019] Preferably, the application includes increasing the contents of acetoin, pulegone, acetophenone, and cyclohexanol in the fermented guava juice beverage and decreasing the contents of caryophyllene, humulene, and hexanal.

[0020] Preferably, the application includes increasing the relative abundances of caffeic acid 4-O-glucuronide, L-xylonic acid, nicotinic acid mononucleotide, and bergenin in the fermented guava juice beverage.

[0021] The beneficial effects of the present invention are as follows:

[0022] 1. The strains used in the present invention are all isolated from Hainan's characteristic tropical fermented foods and the feces of long-lived elderly people, are suitable for using guava juice as the fermentation substrate, and have a low cost.

[0023] 2. After the guava juice of the present invention is fermented by lactobacillus, the inhibitory activity of α-Glu is significantly enhanced, which is beneficial to the development of the functional characteristics of guava juice.

[0024] 3. After the guava juice of the present invention is fermented by lactobacillus, both volatile aroma compounds and metabolites beneficial for reducing blood sugar are significantly increased, which is more conducive to improving the flavor of fermented guava juice and the development of its later functionality. Description of the Drawings

[0025] Figure 1 Effects of different lactobacillus fermentations on the physicochemical properties of guava juice, (A) pH; (B) soluble solids; (C) total polyphenols; (D) total flavonoids.

[0026] Figure 2 Effects of fermentation by different Lactobacillus strains on the in vitro activities of guava juice. (A) ABTS + ; (B) DPPH; (C) CUPRA; (D) FRAP; (E) Inhibitory activity against α-glucosidase.

[0027] Figure 3 Metabolomics analysis of guava juice fermented by different Lactobacillus strains. (A) Indole-3-acetic acid; (B) Dihydroxyacetone; (C) D-Ribose; (D) 6-Hydroxyhexanoic acid; (E) DL-Glutamic acid; (F) Methylmalonic acid; (G) Caffeic acid 4-O-glucuronide; (H) L-Xylonic acid; (I) Nicotinamide mononucleotide; (J) Bergenin. Detailed implementation manners

[0028] To illustrate the present invention more clearly, the present invention will be further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the protection scope of the present invention.

[0029] The present invention aims to explore the effects of four Lactobacillus strains from different sources on the flavor, biological activities and metabolic characteristics of guava juice, and to determine the specificity of the strains by measuring their physicochemical properties, in vitro activities, volatile compounds and metabolites. The technical route of the present invention mainly involves the following aspects:

[0030] I. Select four Lactobacillus strains from different sources (Lactobacillus plantarum HNU082, Lactobacillus plantarum HNU072, Lactobacillus plantarum HNU503 and Lactobacillus fermentum HNU508), inoculate the bacterial solution into pasteurized (85 °C, 15 min) guava juice at 4% (V / V), and ferment at 37 °C for 36 hours to prepare fermented guava juice. Among them:

[0031] Lactobacillus plantarum HNU082 and Lactobacillus plantarum HNU072 were respectively isolated from Hainan specialty fermented foods, fish tea and pickled bamboo shoots, and Lactobacillus plantarum HNU503 and Lactobacillus fermentum HNU508 were isolated from the feces of centenarians in Wanning County, Hainan Province;

[0032] Lactiplantibacillus plantarum HNU082 was deposited at the Guangdong Provincial Culture Collection of Microorganisms (GDMCC) on March 9, 2021, with the deposit number GDMCC NO: 61552. The deposit address is the fifth floor of the Experimental Building of the Guangdong Institute of Microbiology, No. 100, Middle Xianlie Road, Guangzhou, China. The taxonomic name is Lactiplantibacillus plantarum. For reference, see patent application CN113337428A;

[0033] Lactiplantibacillus plantarum HNU072 was deposited at the Guangdong Provincial Culture Collection of Microorganisms (GDMCC) on April 22, 2024, with the deposit number GDMCC NO: 64545. The deposit address is the 5th floor of the Institute of Microbiology, Guangdong Academy of Sciences, Building 59, No. 100 Compound, Middle Xianlie Road, Guangzhou. The taxonomic name is Lactiplantibacillus plantarum;

[0034] Lactiplantibacillus plantarum HNU503 was deposited at the Guangdong Provincial Culture Collection of Microorganisms (GDMCC) on April 22, 2024, with the deposit number GDMCC NO: 64547. The deposit address is the 5th floor of the Institute of Microbiology, Guangdong Academy of Sciences, Building 59, No. 100 Compound, Middle Xianlie Road, Guangzhou. The taxonomic name is Lactiplantibacillus plantarum;

[0035] Limosilactobacillus fermentum HNU508 was deposited at the Guangdong Provincial Culture Collection of Microorganisms (GDMCC) on April 22, 2024, with the deposit number GDMCC NO: 64548. The deposit address is the 5th floor of the Institute of Microbiology, Guangdong Academy of Sciences, Building 59, No. 100 Compound, Middle Xianlie Road, Guangzhou. The taxonomic name is Limosilactobacillus fermentum.

[0036] For simplicity of description, the four strains in the present invention are still simply referred to as "lactobacilli".

[0037] II. Measure the pH, soluble solids, total polyphenols and total flavonoids content of the fermented guava juice;

[0038] III. Measure the antioxidant activities (ABTS + , DPPH, CUPRAC and FRAP) and the inhibitory activity against α-Glu of the fermented guava juice respectively;

[0039] IV. Sensory evaluation of fermented guava juice and determination of changes in volatile metabolites in guava juice before and after fermentation using HS-SPME-GC-MS technology;

[0040] V. Analysis of metabolites in fermented guava juice using LC-MS technology.

[0041] Examples

[0042] I. Preparation of Fermented Guava Juice

[0043] Take the strain stored at -80°C, resuscitate it in MRS medium (peptone 10.0 g / L, beef extract powder 5.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, ammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, Tween 80 1.0 g / L, pH value 5.7 ± 0.2). After subculturing 3 times, centrifuge the strain (4°C, 4000 r / min, 5 min) to collect Lactobacillus, and then resuspend it in sterile normal saline (0.9% NaCl solution) to a concentration of 10^9 CFU / mL. Inoculate the bacterial solution into pasteurized (85°C, 15 min) guava juice (cut the fruits into pieces and squeeze them into juice using conventional methods, no special treatment is required) at 4% (V / V) respectively, and ferment at 37°C for 36 hours to obtain fermented guava juice.

[0044] Among them, the bacterial solution concentration can be 10^8 - 10^9 CFU / mL, the inoculation amount can be 3% - 10% V / V, and the fermentation time can be 24 - 48 hours.

[0045] II. Determination of pH Value and Soluble Solids after Fermentation

[0046] The pH value of the guava juice sample was measured by a calibrated pH meter (PHS-25, Leici, China). The soluble solids content was measured using a digital refractometer (SW-32D, Suwei, China). The contents of total acid and total soluble sugar in the guava juice were detected according to the national standards GB / T 12456-2021 and NY / T2742-2015 respectively.

[0047] The test results showed that the initial pH value of the guava juice was 4.24 ± 0.07, and after fermentation with Lactobacillus, it decreased significantly. Among them, after fermentation with Lactobacillus plantarum HNU072, it decreased to the lowest of 3.24 ± 0.05 ( Figure 1 A). Figure 1 Figure B shows that after fermentation of guava juice by Lactobacillus, the soluble solids decreased to varying degrees. This indicates that Lactobacillus consumed sugar to produce acid during fermentation and successfully fermented the guava juice.

[0048] III. Determination of total phenol and total flavonoid contents

[0049] The total polyphenol and total flavonoid contents were determined by Folin-Ciocalteu colorimetric method and AlCl3 colorimetric method respectively, and the results were expressed as milligrams of gallic acid equivalent (GAE) per gram of sample in dry mass (DM), with the unit of GAE / g DW.

[0050] The determination results showed that lactobacilli also increased the contents of total polyphenols and total flavonoids after fermentation ( Figure 1 C, D). The content of unfermented guava juice was 797.765 ± 9.636 mg GAE / g DW. After fermentation by Lactobacillus plantarum HNU503, the polyphenol content increased to a maximum of 944.235 ± 26.650 mg GAE / g DW. This indicated that lactobacilli fermentation could increase the contents of functional components in guava juice.

[0051] IV. Determination of antioxidant activity

[0052] According to four different antioxidant methods of ABTS + , DPPH, CUPRAC, and FRAR, the antioxidant activity of fermented guava juice was evaluated. Trolox was used as the positive control and a standard curve was plotted. The ABTS + / DPPH scavenging activity value was expressed as μmol Trolox (TE) / g DM sample.

[0053]

[0054] Where: Ac is the absorbance of the mixture of ABTS / DPPH solution and sample solvent, Ai is the absorbance of the mixture of DPPH / ABTS solution and sample solution, and Aj is the absorbance of the mixture of solvent and sample solution.

[0055] For the ferric ion reducing antioxidant power (FRAP) and cupric ion reducing antioxidant capacity (CUPRAC), Trolox was used as the positive control and a standard curve was plotted. FRAP and CUPRAC were expressed as μmol Trolox (TE) / g DW.

[0056] The antioxidant activity was reflected by the values of ABTS + , DPPH, CUPRA, and FRAP. The results showed that after fermentation by lactobacilli, the in vitro antioxidant property of guava juice was improved to varying degrees, and the fermentation effects of Lactobacillus plantarum HNU082 and Lactobacillus plantarum HNU072 were better ( Figure 2 A-D).

[0057] V. α-Glu Inhibition Activity Assay

[0058] Dissolve 100 μL of 1 U / ml α-Glu in 0.1 M phosphate buffer (PBS, pH 6.8), incubate with 100 μL of polyphenol extract at 37 °C for 10 min, then add 100 μL of 5 mM PNPG solution and react at 37 °C for 20 min, and add 500 μL of 1 M Na2CO3 solution to terminate the reaction. Measure the absorbance at 405 nm. Using acarbose and PBS as the positive control and blank control, calculate the α-Glu inhibition activity according to the following formula:

[0059] α-Glu inhibition rate (%) = ((A1 - A0) - (B1 - B0)) / (A1 - A0) × 100%

[0060] Where A1, A0, B1, and B0 represent the absorbances of the blank test group (containing PBS buffer solution and enzyme), blank control group (containing only PBS buffer solution), sample test group (containing guava juice sample, PBS buffer solution and enzyme), and sample control group (containing guava juice sample and PBS buffer solution), respectively.

[0061] Evaluate the inhibitory activity of fermented guava juice on α-Glu through in vitro enzyme inhibition experiment, as Figure 2 shown in Figure E. Guava juice fermented by different Lactobacillus strains all had certain inhibitory effects on α-Glu, among which Lactobacillus plantarum HNU082 (IC 50 = 23.159 ± 1.560 μg / mL) and Lactobacillus plantarum HNU072 (IC 50 = 25.668 ± 2.099 μg / mL) had relatively better inhibitory effects and greater potential to enhance the hypoglycemic activity of guava juice.

[0062] VI. Determination of Volatile Compounds

[0063] HS-SPME-GC-MS was adopted and appropriately modified to identify the changes in volatile metabolites before and after the fermentation of guava juice. 5 mL of the fermented guava juice sample was accurately pipetted into a 20 mL headspace vial and immediately sealed. Immediately afterwards, a headspace gas chromatography-mass spectrometry instrument (7697A-8890-7000D) from Agilent was used to analyze the sample. The specific headspace parameters were as follows: the heating oven temperature was 130 °C, the transfer line temperature was 170 °C, the fermented guava juice was equilibrated for 20 min and then injected, the duration was 0.5 min, and the GC cycle time was 35 min. The detailed chromatographic parameters were as follows: the fermented guava juice was separated by a VF-WAXms capillary column (25 m × 0.25 mm × 0.2 μm, Agilent CP9204) with an injection volume of 1 μL and a split ratio of 10:1, and then entered the mass spectrometry detection. The injection port temperature was 180 °C, high-purity helium was used as the carrier gas, and the flow rate was maintained at 2 mL / min. The temperature was raised from 40 °C (equilibrated for 2 min) to 100 °C and then to 230 °C (run for 7 min). The mass spectrometry parameters were as follows: impact mode (EI, 70 eV), full scan mode (m / z 30 - 1000, 3.2 scans / s). Then the original data was exported, compared with the NIST (2017) database, and after identifying the compounds, the response intensity of the mass spectrometry peaks of the guava juice samples was normalized by the total sum normalization method.

[0064] The results showed that compared with the unfermented guava juice, four Lactobacillus strains produced more aromatic volatile substances, including Acetoin, Pulegone, Acetophenone, and Cyclohexanol. Among them, Acetoin is often used as a flavoring agent and has a unique buttery flavor; Pulegone is the main component of mint and is used to flavor foods and beverages; Acetophenone has the sweetness of orange blossom or jasmine; Cyclohexanol can impart the aroma of mushrooms, baking, and sweet fruits.

[0065] On the other hand, the fermentation of guava juice by four Lactobacillus strains could reduce some olefins and aldehydes, which would produce unpleasant flavors and odors. After fermentation by Lactobacillus, the contents of Caryophyllene, Humulene, and Hexanal in guava juice all decreased (Table 1).

[0066] Table 1 Contents of characteristic volatile compounds in guava juice fermented by different Lactobacillus strains

[0067]

[0068] VII. Determination of non-targeted metabolites

[0069] The UHPLC-Q Exactive HF-X system was adopted. The fermented guava juice was subjected to low-temperature ultrasonic extraction (5°C, 30 min, 40 KHz), then allowed to stand (-20°C, 30 min), centrifuged (4°C, 15 min, 13000 g), and the supernatant was taken for instrumental analysis.

[0070] The chromatographic conditions for LC-MS analysis were as follows: HSS T3 chromatographic column (100 mm × 2.1 mm i.d., 1.8 μm), flow rate of 0.40 mL / min, and column temperature of 40°C. Mobile phase A (containing 0.1% formic acid) was composed of 95% water and 5% acetonitrile, and mobile phase B (containing 0.1% formic acid) was composed of 47.5% acetonitrile, 47.5% isopropanol and 5% water. The mass spectrometric conditions for LC-MS analysis were: positive and negative ion scanning modes, mass scanning range of 70 - 1050 m / z, with the ion spray voltages set at 3500 V and -3500 V respectively. The flow rates of the sheath gas and auxiliary gas (425°C) were 50 psi and 13 psi respectively. The temperature of the ion transfer tube was 325°C, and the normalized collision energy was 20 - 40 - 60 V cyclic collision energy. The resolutions of the first-order and second-order mass spectra were 6000 and 7500 respectively, and data were collected in the DDA mode.

[0071] Data processing was carried out using Progenesis QI (Waters Corporation, Milford, USA) software. Meanwhile, the MS and MSMS mass spectrometric information was matched with the public metabolite databases HMDB (http: / / www.hmdb.ca / ) and Metlin (https: / / metlin.scripps.edu / ) as well as the self-built database of Meiji to obtain metabolite information. The selection of significantly different metabolites was determined based on the variable importance in projection (VIP) values obtained from the OPLS-DA model and the p-values of the student's t-test. Metabolites with VIP > 1 and p < 0.05 were identified as significantly different metabolites.

[0072] The results showed that after fermentation by Lactobacillus, the metabolic profile of guava juice changed significantly ( Figure 3 ). Among them, the main increased metabolites of Lactobacillus from different sources were not completely the same. In particular, after fermentation by four Lactobacillus strains, caffeic acid 4-O-glucuronide, L-xylonic acid, nicotinamide mononucleotide and bergenin were all increased, and these metabolites were all closely related to antioxidant activity, thus improving the antioxidant activity of the fermented guava juice.

[0073] Obviously, the above embodiments of the present invention are merely examples for more clearly illustrating the present invention, rather than limitations on the implementation manners of the present invention. For those of ordinary skill in the art, based on the above description, other different forms of changes or modifications can be made. It is impossible to enumerate all the implementation methods here. Any obvious changes or modifications derived from the technical solutions of the present invention still fall within the protection scope of the present invention.

Claims

1. A guava juice fermented beverage, which uses guava juice as a fermentation substrate and contains fermented mucus lactobacillus ( Limosilactobacillus fermentum ) HNU508 is fermented by a lactobacillus fermentation agent. The fermented mucus lactobacillus HNU508 has been deposited in the Guangdong Microbial Culture Collection Center (GDMCC) on April 22, 2024, with a deposit number of GDMCC NO: 64548. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The guava juice fermented drink according to claim 1, characterized in that, The Lactobacillus starter culture is resuspended in sterile physiological saline to a concentration of 10 8 ~10 9 CFU / mL, the inoculation amount in guava juice is 3% - 10% V / V, and the fermentation conditions are: fermenting at 37 °C for 24 - 48 hours; the guava juice is pasteurized guava juice.

3. Application of Lactobacillus starter containing Limosilactobacillus fermentum HNU508 in improving the flavor and function of guava juice fermented beverage. Limosilactobacillus fermentum HNU508 was deposited at the Guangdong Microbial Culture Collection Center (GDMCC) on April 22, 2024, with the deposit number GDMCC NO: 64548 and the deposit address: 5th Floor, Building 59, Institute of Microbiology, Guangdong Academy of Sciences, No. 100, Xianlie Middle Road, Guangzhou. The application includes increasing the contents of total polyphenols and total flavonoids in the guava juice fermented beverage, increasing the antioxidant activity and α-Glu inhibitory activity of the guava juice fermented beverage, increasing the contents of Acetoin, Pulegone, Acetophenone and Cyclohexanol in the guava juice fermented beverage and decreasing the contents of Caryophyllene, Humulene and Hexanal, and increasing the relative abundances of 4-O-glucuronide of caffeic acid, L-xylonic acid, nicotinamide mononucleotide and bergenin in the guava juice fermented beverage.

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