Beverage in which the off-flavor of the culture medium of lactic acid bacteria is inhibited

Abstract

The objective of this invention is to provide a beverage containing dead lactic acid bacteria, in which the off-flavor of the culture medium for lactic acid bacteria is suppressed, and a method for manufacturing the same. This objective is achieved when the concentration of dead lactic acid bacteria (one or more) at 1.3 billion cells / L or higher, and the concentration of hesperidin at 10–1000 mg / L.

Description

Technical Field

[0001] This invention relates to beverages and methods for manufacturing the same. More specifically, it relates to beverages in which the off-flavors of the lactic acid bacteria culture medium are suppressed by containing dead lactic acid bacteria and hesperidin at a specified concentration. Background Technology

[0002] To meet people's growing health awareness, various beverages containing dead lactic acid bacteria have been launched on the market. However, in beverages containing dead lactic acid bacteria, the off-flavor of the lactic acid bacteria culture medium can sometimes impair the aroma of the beverage.

[0003] As a related technology for suppressing the off-flavor of lactic acid bacteria culture media in beverages, for example, Patent Document 1 discloses the addition of at least one selected from the group consisting of nocacolone (less than 20,000 ppb), linalool (less than 50,000 ppb), and Valencia citriene (less than 50,000 ppb) to a beverage containing lactic acid bacteria powder with a pH greater than 4.6. However, the ability to suppress the off-flavor of lactic acid bacteria culture media in beverages by containing hesperidin at a specified concentration is currently unknown.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent Application Publication No. 2019-103408 Summary of the Invention

[0007] The problem that the invention aims to solve

[0008] Beverages containing dead lactic acid bacteria present the following issues: the off-flavors of the lactic acid bacteria culture medium can sometimes impair the aroma of the beverage.

[0009] The subject of this invention is to provide a beverage containing dead lactic acid bacteria in which the culture medium of lactic acid bacteria is suppressed, and a method for manufacturing the same.

[0010] Methods for solving problems

[0011] In order to solve the above-mentioned problems, the inventors conducted in-depth research on various methods and found that when the concentration of hesperidin in a beverage containing more than 1.3 billion dead cells of one or more lactic acid bacteria is set at 10 to 1000 mg / L, the off-flavor of the lactic acid bacteria culture medium can be suppressed, thus completing the present invention.

[0012] That is, according to the present invention, the following inventions, etc., can be provided.

[0013] (1) A beverage wherein the concentration of dead cells of one or more lactic acid bacteria is more than 1.3 billion / L and the concentration of hesperidin is 10 to 1000 mg / L.

[0014] (2) The beverage described in (1) above has a pH of 2.5 or higher and 8.0 or lower.

[0015] (3) The beverage according to (1) or (2) above, wherein the lactic acid bacteria are selected from one or more of the group consisting of bacteria of the genus Lactococcus.

[0016] (4) A method for manufacturing a beverage, characterized in that, in the manufacturing of the beverage, the beverage contains one or more dead cells of lactic acid bacteria at a concentration of more than 1.3 billion cells / L and contains hesperidin at a concentration of 10 to 1000 mg / L.

[0017] (5) A method for suppressing off-flavors of a culture medium for lactic acid bacteria in a beverage, characterized in that, in the manufacture of the beverage, the beverage contains one or more dead cells of lactic acid bacteria at a concentration of more than 1.3 billion cells / L and contains hesperidin at a concentration of 10 to 1000 mg / L.

[0018] Invention Effects

[0019] According to the present invention, a beverage containing dead lactic acid bacteria culture medium in which off-flavors are suppressed, and a method thereof, etc., can be provided. Attached Figure Description

[0020] Figure 1 A diagram illustrating the relationship between Lactococcus lactis subsp. JCM5805 and its equivalents (strains derived from this strain and strains from which this strain originated). Detailed Implementation

[0021] This invention includes: [1] A beverage (hereinafter also referred to as "the beverage of the present invention"), wherein the concentration of dead cells of one or more lactic acid bacteria is more than 1.3 billion / L, and the concentration of hesperidin is 10 to 1000 mg / L. [2] A method for manufacturing a beverage (hereinafter also referred to as "the manufacturing method of the present invention"), characterized in that, in the manufacturing of the beverage, the beverage contains one or more dead cells of lactic acid bacteria at a concentration of more than 1.3 billion cells / L and contains hesperidin at a concentration of 10 to 1000 mg / L. [3] A method for suppressing off-flavors of a culture medium for lactic acid bacteria in a beverage (hereinafter also referred to as "the suppression method of the present invention"), characterized in that, in the manufacture of the beverage, the beverage contains one or more dead cells of one or more lactic acid bacteria at a concentration of more than 1.3 billion cells / L, and contains hesperidin at a concentration of 10 to 1000 mg / L; etc.

[0022] It should be noted that, unless otherwise stated, the numerical range represented by "~" in this specification naturally includes the values ​​at both ends of "~". Furthermore, although "containing" and "adding" have different meanings, this specification is also considered to describe inventions in which "containing" is replaced with "adding" throughout the entire description of the invention. For example, it is also considered to describe inventions in which phrases such as "containing..." are replaced with "adding...", and inventions in which "makes...containing" is replaced with "adding...".

[0023] (Dead cells of lactic acid bacteria)

[0024] In this invention, one or more dead cells of lactic acid bacteria are used.

[0025] "Lactic acid bacteria" is a general term for all bacteria recognized as lactic acid bacteria in taxonomy, without being limited by genus, species, or strain. As "lactic acid bacteria," examples include bacteria that produce large amounts of lactic acid (preferably more than 50% of the consumed sugar) through lactic fermentation of sugars. Examples include bacteria of the genera *Lactococcus*, *Lactobacillus*, *Streptococcus*, *Leuconostoc*, *Pediococcus*, *Enterococcus*, *Oenococcus*, *Bifidobacterium*, *Weissella*, and *Tetragenococcus*.

[0026] It should be noted that the *Lactobacillus* bacteria in this invention include bacteria that were classified as *Lactobacillus* before the reclassification of the genus. For example, this includes bacteria newly classified as *Acetilactobacillus*, *Agrilactobacillus*, *Amylolactobacillus*, *Apilactobacillus*, *Bombilactobacillus*, *Companilactobacillus*, *Dellaglioa*, *Fructilactobacillus*, *Furfurilactobacillus*, *Holzapfelia*, *Lacticaseibacillus*, *Lactiplantibacillus*, and *Stonewall* bacteria that have been reclassified as *Lactobacillus*. Bacteria belonging to the genera *Lapidilactobacillus*, *Latilactobacillus*, *Lentilactobacillus*, *Levilactobacillus*, *Ligilactobacillus*, *Limosilactobacillus*, *Liquorilactobacillus*, *Loigolactobacillus*, *Paralactobacillus*, *Paucilactobacillus*, *Schleiferilactobacillus*, and *Secundilactobacillus*.

[0027] The genus and species of lactic acid bacteria used in this invention are not particularly limited. Examples include one or more bacteria selected from the group consisting of *Lactococcus*, *Lactobacillus*, *Streptococcus*, *Leuconostoc*, *Pediococcus*, *Enterococcus*, *Sacchariformis*, *Bifidobacterium*, *Weisseria*, and *Tetracoccus*. Preferably, one or more bacteria selected from the group consisting of *Lactococcus*, *Lactobacillus*, and *Pediococcus* are included. More preferably, one or more bacteria selected from the group consisting of *Lactococcus* and *Lactobacillus* are included. Even more preferably, one or more bacteria selected from the group consisting of *Lactococcus* are included. Further preferably, one or more bacteria selected from the group consisting of *Lactococcus lactis* are included. Even more preferably, one or more bacteria selected from the group consisting of *Lactococcus lactis* subsp. *lactococcus* are included.

[0028] As a more specific preferred embodiment of the lactic acid bacteria in this invention, examples selected include *Lactococcus lactis* subsp. *lactis* (e.g., JCM5805, NBRC12007, NRIC1150, JCM20101, JCM7638, ATCC11454), *Lactococcus lactis* subsp. *lactis* biovar. *diacetylactis*, *Lactococcus lactis* subsp. *cremoris* (e.g., JCM16167, NBRC100676), and *Lactococcus raffinose*. *Lactococcus raffinolactis*, *Lactococcus piscium*, *Lactococcus plantarum* (e.g., *Lactococcus plantarum* JCM11056), *Lactococcus garvieae* (e.g., *Lactococcus garvieae* NBRC100934), *Lactococcus lactis subsp. hordniae* (e.g., *Lactococcus lactis subsp. hordniae* JCM1180, *Lactococcus lactis subsp. hordniae* JCM11040), *Lactobacillus acidophilus* (e.g., *Lactobacillus acidophilus* L-92), *Lactobacillus delbrueckii subsp. bulgaricus* (e.g., *Lactobacillus bulgaricus* OLL1073R-1), and *Lactobacillus delbrueckii subsp.* Lactobacillus delbrueckii and Lactobacillus delbrueckii subsp.Lactobacillus, Lactobacillus casei, Lactobacillus paracasei (including Lactobacillus paracasei KW3110 and Lactobacillus paracasei MCC1849, etc.), Lactobacillus gasseri (including Lactobacillus gasseri SBT2055, etc.), Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus parakefiri (including Lactobacillus parakefiri JCM8573, etc.), Lactobacillus plantarum (including Lactobacillus plantarum L-137, etc.), Lactobacillus brevis, Lactobacillus rhamnosus (including Lactobacillus rhamnosus GG and Lactobacillus rhamnosus CRL1505, etc.), Lactobacillus pentosaccharide Lactobacillus pentosus (ONRICb0240, etc.), Lactobacillus fermentum, Lactobacillus fructivorans, Lactobacillus hilgardii, Streptococcus salivarius subsp. thermophilus, and Leuconostoc mesenteroides subsp.*Leuconostoc lactis* (e.g., Leuconostoc lactis NBRC12455), *Leuconostoc carnosum* (e.g., Leuconostoc carnosum JCM9695), *Pediococcus damnosus* (e.g., Pediococcus damnosus JCM5886), *Pediococcus pentosaceus*, *Pediococcus acidilactici* (e.g., Pediococcus acidilactici JCM8797 and Pediococcus K15), *Pediococcus cellicola*, *Pediococcus claussenii*, *Pediococcus ethanolidurans*, *Pediococcus inopinatus*, *Pediococcus parvulus*, *Pediococcus stearothermia* *Bifidobacterium stilesii*, *Enterococcus faecalis*, *Enterococcus faecium*, *Enterococcus alcedinis*, *Oenococcus oeni* (e.g., *Oenococcus oeni* JCM6125), *Bifidobacterium animalis subsp. lactis* (e.g., *Bifidobacterium animalis subsp. lactis* JCM10602), and *Bifidobacterium longum subsp.*One or more bacteria selected from the group consisting of *Bifidobacterium infantis* (e.g., *Bifidobacterium longum* subsp. *infancier* JCM1222), *Weissella paramesenteroides* (e.g., *Weissella paramesenteroides* JCM9890), *Weissella viridescens* (e.g., *Weissella viridescens* JCM1174), and *Tetragenococcus halophilus* (e.g., *Tetragenococcus halophilus* NRIC0098), preferably including *Lactococcus lactis* subsp. *lactolaccos*, *Lactococcus lactis* diacetyl, *Lactococcus lactis* subsp. *lactolaccos* fat, *Lactococcus raffinis*, *Lactococcus spp.*, *Lactococcus plantarum*, *Lactococcus gasseri*, *Lactococcus lactis* subsp. *holygrenella*, *Lactobacillus acidophilus*, *Lactobacillus delbrueckii* subsp. *bulgaricus*, and *Lactobacillus delbrueckii* delbrueckii. One or more bacteria from the group consisting of *Lactobacillus delbrueckii* subspecies, *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus johnsonii*, *Lactobacillus causativeis*, *Lactobacillus plantarum*, *Lactobacillus brevis*, *Lactobacillus rhamnosus*, *Lactobacillus pentosus*, *Lactobacillus fermentum*, *Lactobacillus fructose*, *Lactobacillus hessei*, harmful *Pediococcus*, *Pediococcus pentosus*, *Pediococcus lactis*, *Pediococcus cellarus*, *Pediococcus clavatum*, ethanol-resistant *Pediococcus*, accidental *Pediococcus*, small *Pediococcus*, and *Pediococcus stearothermiae*, more preferably selected from *Lactococcus lactis* subspecies and *Lactococcus lactis* subspecies diacetyl... The following bacteria are listed: *Lactococcus lactis* subsp. *milk fat*, *Lactococcus raffinis*, *Lactococcus spp.*, *Lactococcus plantarum*, *Lactococcus gasseri*, *Lactococcus lactis* subsp. *Horacetamella*, *Lactobacillus acidophilus*, *Lactobacillus delbrueckii* subsp. *bulgaricus*, *Lactobacillus delbrueckii* subsp. *delbrueckii*, *Lactobacillus delbrueckii* subsp. *delbrueckii*, *Lactobacillus casei*, *Lactobacillus paracasei*, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus johnsonii*, *Lactobacillus caudatus*, *Lactobacillus plantarum*, *Lactobacillus brevis*, *Lactobacillus rhamnosus* (Lactobacillus rhamnosus GG and Lactobacillus rhamnosus CRL1505), *Lactobacillus pentosus*, *Lactobacillus fermentum*, *Lactobacillus fructose*, and *Lactobacillus hesperidin*. The group contains one or more bacteria, more preferably one or more bacteria selected from the group consisting of *Lactococcus lactis* subsp. *lactococcus*, more preferably one or more bacteria selected from the group consisting of *Lactococcus lactis* subsp. *lactococcus* JCM5805, *Lactococcus lactis* subsp. *lactococcus* JCM20101, *Lactococcus lactis* subsp. *lactococcus* subsp. *lactococcus* NRIC1150, *Lactococcus lactis* subsp. *lactococcus* JCM7638, and *Lactococcus lactis* subsp. *lactococcus* ATCC11454, and particularly preferably *Lactococcus lactis* subsp. *lactococcus* JCM5805.

[0029] Furthermore, as a further preferred embodiment of the genus and species of lactic acid bacteria used in the present invention, one or more species selected from the group consisting of *Lactococcus* bacteria can be listed, preferably one or more species selected from the group consisting of *Lactococcus lactis* subsp. *milk* JCM5805, *Lactococcus lactis* subsp. *milk* NBRC12007, *Lactococcus lactis* subsp. *milk* NRIC1150, *Lactococcus lactis* subsp. *milk* JCM20101, *Lactococcus lactis* subsp. *milk* JCM7638, *Lactococcus lactis* subsp. *milk* ATCC11454, *Lactococcus gasseri* NBRC100934, *Lactococcus lactis* subsp. *milk fat* JCM16167, *Lactococcus lactis* subsp. *milk fat* NBRC100676, *Lactococcus lactis* subsp. *Hosne* JCM1180, and *Lactococcus lactis* subsp. *Hosne* JCM11040.

[0030] Furthermore, as one embodiment of the lactic acid bacteria in this invention, preferably selected lactic acid bacteria other than *Lactobacillus paracasei* MCC1849, *Lactobacillus* bacteria other than *Lactobacillus paracasei* MCC1849, lactic acid bacteria other than *Lactobacillus paracasei*, *Lactobacillus* bacteria other than *Lactobacillus*, and lactic acid bacteria other than *Lactobacillus*. Among the *Lactobacillus* bacteria other than *Lactobacillus paracasei*, preferably selected are *Lactobacillus acidophilus*, *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus delbrueckii* subsp. delbrueckii, *Lactobacillus delbrueckii* subsp. lactobacillus, *Lactobacillus casei*, *Lactobacillus gasseri*, *Lactobacillus helveticus*, *Lactobacillus johnsonii*, *Lactobacillus caudatus*, *Lactobacillus plantarum*, *Lactobacillus brevis*, *Lactobacillus rhamnosus*, *Lactobacillus pentosus*, *Lactobacillus fermentum*, *Lactobacillus fructose*, and *Lactobacillus hesperidin*.

[0031] Regarding the lactic acid bacteria strains listed in this specification, in this invention, when a specified beverage contains dead lactic acid bacteria cells and hesperidin at a specified concentration, strains equivalent to the aforementioned strains are also included, provided they can suppress the off-flavor of the lactic acid bacteria culture medium in the beverage. Here, equivalent strains refer to strains derived from the aforementioned strains, strains from which the aforementioned strains originated, or descendant strains of the aforementioned strains. Equivalent strains are sometimes also preserved in other strain preservation facilities. Figure 1 This shows strains derived from *Lactococcus lactis* subsp. *lactococcus* JCM5805 and the strains from which *Lactococcus lactis* subsp. *lactococcus* JCM5805 originates. For Figure 1 Equivalent strains of *Lactococcus lactis* subsp. *lactocopherol* JCM5805 described herein can be used as the lactic acid bacteria of this invention, provided that they can suppress the off-flavor of the lactic acid bacteria culture medium in a beverage containing dead lactic acid bacteria and hesperidin at a specified concentration. In this specification, the reference to *Lactococcus lactis* subsp. *lactocopherol* JCM5805 also includes equivalent strains of these strains. Similarly, the reference to *Lactobacillus rhamnosus* CRL1505 in this specification also includes equivalent strains of these strains.

[0032] In addition, among the aforementioned lactic acid bacteria strains, the JCM strain can be obtained from the Microbial Materials Development Laboratory of the Bioresource Center at RIKEN (3-1-1 Takanodai, Tsukuba City, Ibaraki Prefecture, Japan), the NBRC strain can be obtained from the Biogenetic Resources Division of the Technical Base for Product Evaluation (2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan), the NRIC strain can be obtained from the Tokyo University of Agriculture Strains Preservation Room (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, Japan), and the ATCC strain can be obtained from the American Type Culture Collection (USA).

[0033] As mentioned above, the *Lactococcus lactis* subsp. *lactococcus* JCM5805 strain can be obtained from the Microbial Materials Development Laboratory of the RIKEN Center for Biological Resources. In this invention, the same strain of JCM5805 preserved in a depository other than the Microbial Materials Development Laboratory of the RIKEN Center for Biological Resources can be used. Specifically, the same strain of JCM5805 can be obtained from the Biogenetic Resources Division of the Technical Base for Product Evaluation (2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan), the Tokyo University of Agriculture Strains Preservation Room (1-1-1 Sakuragaoka, Setagaya-ku, Tokyo, Japan), and the American Culture Collection (United States).

[0034] In this specification, "lactic acid bacteria" refers to dead lactic acid bacteria. The dead lactic acid bacteria used in this invention can be either dried or undried. From the viewpoint of preserving the stability of dead lactic acid bacteria, dried products are preferred, and examples include dried powder.

[0035] There are no particular restrictions on the preparation methods for dead lactic acid bacteria. Examples include: sterilizing the culture medium after culturing lactic acid bacteria and then collecting the bacteria by filtration, centrifugation, etc.; collecting the bacteria from the culture medium after culturing lactic acid bacteria by filtration, centrifugation, etc. and then sterilizing them; etc. If necessary, further drying or crushing treatment can be carried out.

[0036] It should be noted that there are no particular restrictions on sterilization methods; not only heating can be used, but also conventional methods for killing bacteria, such as ultraviolet light and gamma ray irradiation. Alternatively, live lactic acid bacteria can be used, which are then killed during the sterilization process in beverage preparation, resulting in dead lactic acid bacteria.

[0037] In this invention, the concentration of dead cells of one or more lactic acid bacteria in the beverage is not particularly limited as long as it is 1.3 billion cells / L or more. From the viewpoint that the culture medium of lactic acid bacteria, which is the subject of this invention, will have an increased off-flavor and thus make this invention more significant, examples of concentrations of 5 billion cells / L or more, 10 billion cells / L or more, 30 billion cells / L or more are possible, preferably 50 billion cells / L or more, 80 billion cells / L or more, 100 billion cells / L or more, and more preferably 200 billion cells / L or more, 300 billion cells / L or more, or 600 billion cells / L or more. Furthermore, there is no particular upper limit to the concentration of dead lactic acid bacteria in beverages, which can be one or more types of bacteria. For example, from the perspective of minimizing the off-flavor of the lactic acid bacteria culture medium, the total number of dead lactic acid bacteria can be listed as below 6 trillion CFU / L, below 3 trillion CFU / L, below 2 trillion CFU / L, below 1 trillion CFU / L, below 800 billion CFU / L, below 600 billion CFU / L, below 300 billion CFU / L, below 200 billion CFU / L, below 100 billion CFU / L, below 80 billion CFU / L, below 50 billion CFU / L, below 30 billion CFU / L, and below 10 billion CFU / L. These lower and upper limits can be combined arbitrarily.

[0038] The concentration of dead lactic acid bacteria in a beverage can be adjusted, for example, by adjusting the amount of dead lactic acid bacteria incorporated into the beverage.

[0039] It should be noted that, as a method for determining the number of dead lactic acid bacteria in beverages, there are no particular limitations on the list of known methods for determining the number of lactic acid bacteria, such as direct microscopy, particle induction zone method, PCR method, or flow cytometry, with flow cytometry being the preferred method.

[0040] (Hesperidin)

[0041] Hesperidin is a polyphenol found in the peel, pith, segments, and pulp of citrus fruits, etc. There are no particular limitations on the hesperidin used in this invention. Besides hesperidin extracted and / or purified from citrus fruits, hesperidin used as a food additive, juices or processed juices containing hesperidin from citrus fruits, etc., can be used individually or in combination as appropriate. The types of citrus fruits are not particularly limited, and examples include lemon, lime, orange, tangerine, grapefruit, pomelo, iyokan, summer orange, hachisaku mandarin, ponkan, flat lemon, and stinky orange, with orange and tangerine being preferred. Furthermore, in this invention, one or more of hesperidin and hesperidin derivatives can be used as the hesperidin. There are no particular limitations on the hesperidin derivatives; from the viewpoint of higher solubility in water and the ability to contain more hesperidin in beverages, examples include methyl hesperidin and hesperidin glycosides (glycosyltransfer hesperidin). Examples of hesperidin glycosides include glycosylhesperidin (more preferably monoglucosylhesperidin).

[0042] The concentration of hesperidin in this invention (the concentration in the beverage or the added concentration in the beverage) is not particularly limited as long as it is 10 to 1000 mg / L relative to the total amount of the beverage. From the viewpoint of further suppressing the off-odor of the culture medium of lactic acid bacteria, which is the subject of this invention, the lower limit can be 10 mg / L or more, 12 mg / L or more, preferably 15 mg / L or more, 20 mg / L or more, 30 mg / L or more, 40 mg / L or more, more preferably 50 mg / L or more, 60 mg / L or more, 70 mg / L or more, 80 mg / L or more, 90 mg / L or more, and even more preferably 100 mg / L or more. In addition, from the viewpoint that the off-odor of hesperidin itself will not be a problem, the upper limit can be less than 1500 mg / L, preferably less than 1000 mg / L. Other possible upper limits for hesperidin include 900 mg / L and below, 800 mg / L and below, 700 mg / L and below, 600 mg / L and below, 500 mg / L and below, 400 mg / L and below, 300 mg / L and below, 200 mg / L and below, 100 mg / L and below, etc. These lower and upper limits can be combined arbitrarily. Therefore, considering a balance, 10–1000 mg / L, 15–1000 mg / L, preferably 50–1000 mg / L, 50–800 mg / L, 50–500 mg / L, etc., can be listed. It should be noted that the hesperidin concentration in this invention represents the concentration of the total amount of hesperidin contained in the beverage and the amount of hesperidin derivatives converted into hesperidin relative to the overall beverage.

[0043] As one embodiment of the beverage of the present invention, a beverage containing hesperidin at a predetermined concentration as specified in the present invention can be listed. This is achieved by adding fruit juice or processed fruit juice containing hesperidin, such as citrus fruits. Furthermore, as another embodiment of the beverage of the present invention, a beverage containing hesperidin at a predetermined concentration as specified in the present invention can be preferably listed. This is achieved by adding hesperidin in addition to fruit juice or processed fruit juice containing hesperidin, such as citrus fruits.

[0044] It should be noted that the concentration of hesperidin can be calculated and / or determined using methods known to those skilled in the art. For example, analytical methods using HPLC (high performance liquid chromatography) can be cited. More specific examples of methods for determining hesperidin concentration are described below.

[0045] Add 3 mL of water and 30 mL of methanol to 4 g of the sample, and extract by ultrasonic irradiation for 5 minutes. Then, bring the volume to 50 mL. After appropriate dilution, a sample solution can be prepared. High-performance liquid chromatography (HPLC) can be performed under the following conditions.

[0046] [High Performance Liquid Chromatography Operating Conditions] Detector: UV-Vis spectrophotometer Column: XBridge C18, Φ4.6mm×150mm, particle size 3.5μm Column temperature: 40℃ Mobile phase: a mixture of water, acetonitrile, and phosphoric acid (850:150:1) Flow rate: 1.0 mL / min Measurement wavelength: 280nm (pH) The pH of the beverage of this invention is not particularly limited, and examples include 2.5 and above, 2.8 and above, 3.0 and above, 3.2 and above, 3.5 and above, 3.8 and above, and 4.0 and above; and examples include 8.0 and below, 7.0 and below, 6.0 and below, 5.0 and below, 4.6 and below, 4.3 and below, 4.0 and below, 3.8 and below, 3.5 and below, 3.2 and below, and 3.0 and below. More specifically, examples include 2.5 to 8.0, 2.5 to 7.0, and 2.5 to 4.6. The pH value is a measurement value at 20°C.

[0047] The pH of the beverage of the present invention can be adjusted by conventional methods such as adding acidulants and pH adjusters. Examples of pH adjusters include substances that exhibit alkalinity or acidity when dissolved in water, specifically including baking soda (sodium bicarbonate), sodium carbonate, potassium carbonate, sodium hydroxide, potassium hydroxide, sodium phosphate, potassium phosphate, sodium citrate, and carbon dioxide.

[0048] The pH of the beverage of the present invention can be measured using a pH meter or other conventional methods.

[0049] (The beverage of this invention)

[0050] As a beverage of the present invention, there are no particular limitations as long as the concentration of dead cells of one or more lactic acid bacteria is 1.3 billion / L or more and the concentration of hesperidin is 10 to 1000 mg / L.

[0051] The beverage of the present invention, except that the concentration of dead cells of one or more lactic acid bacteria is more than 1.3 billion / L and the concentration of hesperidin is 10 to 1000 mg / L, is not particularly different from ordinary "beverages" in terms of raw materials, manufacturing methods and manufacturing conditions.

[0052] The beverage of the present invention contains at least 1.3 billion / L of dead cells of one or more lactic acid bacteria and 10-1000 mg / L of hesperidin as essential components. The beverage of the present invention may contain optional components within a range that does not impair the effects of the present invention. These optional components may include one or more selected from the group consisting of acidulants, sweeteners, bittering agents, flavorings, colorings, fruit juice or processed fruit juice, vegetable juice or processed vegetable juice, antioxidants, preservatives, stabilizers (thickening stabilizers, etc.), milk components, soy components, lactic acid fermentation products, emulsifiers, pH adjusters, dietary fiber, vitamins, and minerals, or may not contain any of these components.

[0053] Examples of acidulants include phosphoric acid, lactic acid, tartaric acid, citric acid, malic acid, and adipic acid. Examples of sweeteners include sucrose, fructose, glucose, maltose, starch saccharides, reduced starch syrup, dextrin, cyclodextrin, trehalose, brown sugar, and honey; sugar alcohols such as sorbitol, erythritol, xylitol, and mannitol; and high-sweetness sweeteners such as sucralose, stevia, acesulfame potassium, sodium saccharin, aspartame, glycyrrhizic acid, dipotassium glycyrrhizate, sematrandez, and neotame.

[0054] In this instruction manual, "juice" refers to the juice extracted from a fruit. Juice can be produced using methods commonly used to manufacture fruit beverages, such as pressing the whole fruit (including the peel) using an in-line juicer to separate the juice from the pulp and then sterilizing and cooling the extracted juice; or separating the whole fruit into the peel and pulp, or cutting the fruit in half and extracting only the pulp, followed by sterilization and cooling. Juice includes concentrated juice obtained by concentrating the juice extracted from the fruit, and reconstituted juice obtained by diluting the concentrated juice. Fruit juicing refers to the process of crushing the fruit and then juicing or filtering it, removing the peel, seeds, etc., to obtain fruit puree or similar products.

[0055] Fruit juice can be cloudy or clear after clarification. Methods for clarifying fruit juice include enzymatic treatment, fine filtration, and ultrafiltration.

[0056] As the juice, commercially available juices, concentrated juices, jams, etc., can be used. Specifically, juices and concentrated juices specified in the JAS standard (Japanese Agricultural and Forestry Standard for Fruit Beverages) can be used, for example, one or more of these can be used to prepare the beverage of the present invention.

[0057] In this specification, "processed fruit juice products" refers to products made from processed fruit juice. Processed fruit juice products include, for example, fruit juice extracts and fruit juice flavorings.

[0058] The beverage of the present invention can be manufactured by any stage of a conventional manufacturing method of "beverage" known in the art, containing dead cells of one or more lactic acid bacteria at a concentration of more than 1.3 billion cells / L and containing hesperidin at a concentration of 10 to 1000 mg / L.

[0059] The beverage of the present invention may not be packaged in a container, but it is preferred to package the beverage in a container. Examples of containers include resin bottles such as PET bottles, polypropylene bottles, and polyvinyl chloride bottles; bottle containers; can containers, etc.

[0060] The beverage of the present invention can be heat-sterilized or not. Compared to the case without heating, by heating a beverage containing a specified concentration of dead lactic acid bacteria (e.g., dried powder of lactic acid bacteria) at a specified high temperature (e.g., 65°C or higher, 70°C or higher, 75°C or higher, 80°C or higher, 85°C or higher, 100°C or higher, 120°C or higher, etc.) for a specified time (e.g., 4 minutes or higher, 10 minutes or higher, 20 minutes or higher, 30 minutes or higher, etc.), the off-flavor of the lactic acid bacteria culture medium in the beverage becomes stronger. Therefore, by heat-sterilizing the beverage, the off-flavor of the lactic acid bacteria culture medium, which is the subject of the present invention, is increased, further enhancing the significance of the present invention. Therefore, beverages subjected to heat sterilization are more preferably listed in the present invention. Furthermore, from the viewpoint of improved shelf life, beverages subjected to heat sterilization are more preferably listed in the present invention. Conventional methods and conditions used for beverages such as those packaged in containers can be used as methods and conditions for heat sterilization. Examples of heating temperature and time conditions for sterilizing the beverage of the present invention include, for example, 65°C for 10 minutes or more, 80°C for 10 minutes or more, 85°C for 10 minutes or more, 85°C for 30 minutes or more, 120°C for 4 minutes or more, or conditions with equivalent or higher effectiveness to these conditions.

[0061] The beverage of the present invention can be a beverage sold in any of the following states: refrigerated (0-10°C), at room temperature, or heated (50-70°C), and beverages other than chilled beverages sold in a refrigerated state can be listed.

[0062] There are no particular limitations on the type of beverage of the present invention. Fruit juice beverages containing fruit juice or processed fruit juice products are preferred, more preferably fruit juice beverages containing citrus fruit juice or processed fruit juice products, and most preferably fruit juice beverages containing orange or tangerine juice or processed fruit juice products. Furthermore, beverages of the present invention may include beverages other than fermented milk or lactic acid bacteria beverages.

[0063] (Manufacturing method of the present invention)

[0064] Except for the fact that the beverage contains one or more dead lactic acid bacteria at a concentration of more than 1.3 billion cells / L and hesperidin at a concentration of 10 to 1000 mg / L, the beverage of the present invention can be manufactured according to conventionally known beverage manufacturing methods.

[0065] As a manufacturing method of the present invention, there are no particular limitations on the manufacturing method of the beverage as long as it is characterized by containing one or more dead cells of lactic acid bacteria at a concentration of 1.3 billion cells / L or more and containing hesperidin at a concentration of 10 to 1000 mg / L.

[0066] As a method for making a beverage contain dead cells of one or more lactic acid bacteria at a concentration of 1.3 billion CFU / L or more, and hesperidin at a concentration of 10 to 1000 mg / L, more specifically, a method can be listed in which the raw materials for manufacturing the beverage (e.g., "water" or "water that also contains a portion or all of optional components") (hereinafter collectively referred to as "water, etc.") contain dead cells of one or more lactic acid bacteria and hesperidin, and the concentration of dead cells of one or more lactic acid bacteria is 1.3 billion CFU / L or more (preferably prepared to be 1.3 billion CFU / L or more), and the concentration of hesperidin is 10 to 1000 mg / L (preferably prepared to be 10 to 1000 mg / L). Alternatively, a method can be listed in which dead cells of one or more lactic acid bacteria and hesperidin are simultaneously contained in water, etc., at a specified concentration and as a portion or all of optional components.

[0067] In the manufacturing method of this invention, the beverage contains 1.3 billion / L of dead cells of one or more types of lactic acid bacteria and 10-1000 mg / L of hesperidin as essential components. As a manufacturing method of this invention, the beverage may contain one or more of the following optional components: acidulants, sweeteners, bittering agents, flavorings, colorings, fruit juice or processed fruit juice, vegetable juice or processed vegetable juice, antioxidants, preservatives, stabilizers (thickening stabilizers, etc.), milk components, soybean components, lactic acid fermentation products, emulsifiers, pH adjusters, dietary fiber, vitamins, and minerals; or it may not contain any of these components.

[0068] In the manufacturing method of this invention, there are no particular restrictions on the order in which the manufacturing ingredients are contained, as long as it is possible to manufacture a beverage of this invention containing the manufacturing ingredients used. When making the beverage of this invention into a container, it can be manufactured by filling the container with the liquid formed by mixing the manufacturing ingredients and then sealing it.

[0069] In the manufacturing method of the present invention, heat sterilization of the beverage may or may not be performed. However, as mentioned above, heat sterilization of the beverage increases the off-flavor of the culture medium for the lactic acid bacteria, which is the subject of the present invention, and thus enhances the significance of the present invention. Therefore, in the manufacturing method of the present invention, a method including the step of heat sterilization of the beverage is more preferred. Furthermore, from the viewpoint of improving shelf life, in the manufacturing method of the present invention, a method including the step of heat sterilization of the beverage is more preferred. There are no particular limitations on the heat sterilization method; examples include, for instance, high-temperature short-time sterilization (HTST), pasteurization, ultra-high temperature heat treatment (UHT), and cooking sterilization.

[0070] (The suppression method of the present invention)

[0071] As for the suppression method of the present invention, there are no particular limitations as long as it is a method for suppressing off-flavors of the culture medium of lactic acid bacteria in a beverage that contains one or more dead cells of lactic acid bacteria at a concentration of 1.3 billion cells / L or more and hesperidin at a concentration of 10 to 1000 mg / L.

[0072] In the manufacture of beverages, the method for containing one or more dead cells of lactic acid bacteria at a concentration of 1.3 billion cells / L or more and hesperidin at a concentration of 10 to 1000 mg / L can be the same as the method described in the above-described (manufacturing method of the present invention).

[0073] (Suppression of off-odors in the culture medium of lactic acid bacteria in this invention)

[0074] The beverage of the present invention is a beverage in which the off-flavor of the lactic acid bacteria culture medium is suppressed. In this specification, "culture medium off-flavor" refers to the unpleasant odor originating from the culture medium in which the lactic acid bacteria are cultured. This odor can be problematic, for example, when combined with dead cells (e.g., dried powder) of lactic acid bacteria cultured in a medium containing yeast extract, meat extract, peptone, etc. The off-flavor of the lactic acid bacteria culture medium is a unique aroma accompanied by off-flavors such as umami or animal odors, which is particularly undesirable for typical soft drinks. As described above, the off-flavor of the lactic acid bacteria culture medium, which is the subject of this invention, is increased by the heat sterilization treatment of the beverage, thus making the present invention more significant. Therefore, the suppression method of the present invention is more preferably used for beverages whose manufacturing process includes a heat sterilization treatment.

[0075] In this invention, a beverage in which "the off-flavor of the lactic acid bacteria culture medium is suppressed" refers to a beverage manufactured using the same raw materials and the same manufacturing method at the same final concentration as a beverage containing dead cells of one or more lactic acid bacteria at a concentration of 1.3 billion CFU / L or higher, except that it does not contain hesperidin at a concentration of 10–1000 mg / L (hereinafter also referred to as "control beverage").

[0076] The degree of off-flavor of the lactic acid bacteria culture medium in a beverage, and how that degree of off-flavor compares to the control beverage of the present invention (e.g., whether the off-flavor of the lactic acid bacteria culture medium is suppressed, and to what extent it is suppressed), can be easily and clearly determined by a trained reviewer.

[0077] The invention will be specifically described based on the following examples, but the invention is not limited to these examples.

[0078] Example

[0079] [Experiment 1] The effects of containing hesperidin

[0080] The following experiment was conducted to investigate the effect of hesperidin in beverages on the off-flavor of lactic acid bacteria culture media.

[0081] (1. Preparation of sample beverage)

[0082] To achieve the concentrations of *Lactococcus lactis* subsp. *lactocortisone* JCM5805, hesperidin, and pH specified in Table 2, deionized water was infused with dried dead bacterial cell powder of *Lactococcus lactis* subsp. *lactocortisone* JCM5805, hesperidin, and phosphate, thereby preparing sample beverages for Test Examples 1 to 12. Each sample beverage was filled into a container and sterilized by heating at 80°C for 10 minutes.

[0083] (2. Sensory evaluation test)

[0084] For the degree of culture medium off-odor in the beverage samples from Test Examples 1 to 12, four trained professional reviewers conducted sensory evaluation tests according to the evaluation criteria in Table 1 below. It should be noted that the differences between 1 and 1.5 points, 1.5 and 2 points, 2 and 2.5 points, 2.5 and 3 points, 3 and 3.5 points, 3.5 and 4 points, 4 and 4.5 points, and 4.5 and 5 points are considered to be of the same degree. Furthermore, for each beverage sample, the average of the evaluation results from the four professional reviewers was calculated, rounded to two decimal places, and used as the evaluation result for that beverage sample. The standard deviation was always below 0.5. Beverages with a score reduction of 0.5 or more compared to Test Example 1 (control beverage) can be evaluated as beverages with suppressed culture medium off-odor.

[0085] [Table 1]

[0086] The results of the sensory evaluation tests of the sample beverages in Test Examples 1 to 12 are shown in Table 2.

[0087] [Table 2]

[0088] As shown in Table 2, the off-odors of the culture medium in Test Examples 3-12 were suppressed compared to Test Example 1 (control beverage). This indicates that when a beverage containing dead lactic acid bacteria cells contains hesperidin at a concentration of 10 mg / L or higher, an inhibitory effect on the off-odors of the lactic acid bacteria culture medium can be achieved. Furthermore, this effect increases with the concentration of hesperidin; from the viewpoint of obtaining a greater inhibitory effect on the off-odors of the lactic acid bacteria culture medium, the concentration of hesperidin is preferably 15 mg / L or higher, more preferably 50 mg / L or higher. However, regarding Test Example 12, although the inhibitory effect on the off-odors of the lactic acid bacteria culture medium was high, the strong odor of hesperidin itself was also noted. Therefore, the concentration of hesperidin is preferably less than 1500 mg / L, more preferably less than 1000 mg / L.

[0089] [Experiment 2] The effect of different concentrations of dead lactic acid bacteria

[0090] The following experiment was conducted to investigate the effect of different concentrations of dead lactic acid bacteria on the inhibitory effect of hesperidin on the off-flavor of lactic acid bacteria culture media.

[0091] (1. Preparation of sample beverage)

[0092] The concentrations of *Lactococcus lactis* subsp. *lactocopherol* JCM5805 and hesperidin were set to the values ​​recorded in Table 3. Otherwise, the sample beverages of Test Examples 13–24 were prepared and heat-sterilized using the same method as described in [Test 1]. It should be noted that the sample beverages of Test Examples 1 and 6 were prepared and heat-sterilized using the same method as described in [Test 1].

[0093] (2. Sensory evaluation test)

[0094] For the beverage samples obtained in Test Examples 1, 6, and 13–24, sensory evaluation tests were conducted using the same method as in [Test 1] described above. The results of these tests are shown in Table 3.

[0095] [Table 3]

[0096] The results in Table 3 show that an off-flavor is produced when the concentration of dead lactic acid bacteria exceeds 1.3 billion CFU / L, and the off-flavor intensifies with increasing concentration of dead lactic acid bacteria. Furthermore, regardless of the concentration of dead lactic acid bacteria, the presence of hesperidin in the beverage effectively inhibits the off-flavor of the culture medium.

[0097] [Experiment 3] The effects of different pH levels

[0098] The following experiment was conducted to investigate how different pH levels of beverages affect the inhibitory effect of hesperidin on off-flavors in lactic acid bacteria culture media.

[0099] (1. Preparation of sample beverage)

[0100] The pH was set to the value recorded in Table 4, and the sample beverages of Test Examples 25-34 were prepared and heat-sterilized using the same method as in [Test 1] described above. It should be noted that the sample beverages of Test Examples 1 and 6 were prepared and heat-sterilized using the same method as in [Test 1] described above.

[0101] (2. Sensory evaluation test)

[0102] For the beverage samples obtained in Test Examples 1, 6, and 25–34, sensory evaluation tests were conducted using the same method as in [Test 1] described above. The results are shown in Table 4.

[0103] [Table 4]

[0104] As shown in Table 4, when the pH of the sample beverage is 2.5-8, the presence of hesperidin can inhibit the off-flavor of the culture medium.

[0105] [Experiment 4] Example of a formula when using citrus juice

[0106] The following experiment was conducted using juices from citrus fruits containing hesperidin.

[0107] (1. Preparation of sample beverage)

[0108] 2% orange juice containing hesperidin at a concentration of 355 mg / L was added, resulting in a hesperidin content of 7.1 mg / L from the orange juice. Further addition of hesperidin was made as needed, resulting in a hesperidin content at the concentrations listed in Table 5. Otherwise, the sample beverages of Test Examples 35–42 were prepared and heat-sterilized using the same method as in [Test 1] described above. It should be noted that the sample beverage of Test Example 1 was prepared and heat-sterilized using the same method as in [Test 1] described above.

[0109] It should be noted that the concentration of hesperidin in orange juice was determined using the following method.

[0110] Add 3 mL of water and 30 mL of methanol to 4 g of the analyte, and extract by ultrasonic irradiation for 5 minutes. Then, bring the volume to 50 mL. Dilute appropriately to prepare the sample solution. Perform high-performance liquid chromatography (HPLC) under the following conditions.

[0111] [High Performance Liquid Chromatography Operating Conditions] Detector: UV-Vis spectrophotometer Column: XBridge C18, Φ4.6mm×150mm, particle size 3.5μm Column temperature: 40℃ Mobile phase: a mixture of water, acetonitrile, and phosphoric acid (850:150:1) Flow rate: 1.0 mL / min Measurement wavelength: 280nm (2. Sensory evaluation test) For the beverage samples obtained in Test Examples 1 and 35–42, sensory evaluation tests were conducted using the same method as in [Test 1] described above. The results are shown in Table 5.

[0112] [Table 5]

[0113] As shown in Table 5, when a portion of orange juice is used to bring the hesperidin concentration in the beverage to the specified level, the same effect of inhibiting off-flavors in the culture medium of lactic acid bacteria is achieved as when no orange juice is used (Table 2).

[0114] Industrial availability

[0115] According to the present invention, a beverage containing dead lactic acid bacteria culture medium in which off-flavors are suppressed, and a method thereof, etc., can be provided.

Claims

1. A beverage, wherein, The concentration of dead cells of one or more lactic acid bacteria is above 1.3 billion / L, and the concentration of hesperidin is 10-1000 mg / L.

2. The beverage according to claim 1, wherein the pH is above 2.5 and below 8.

0.

3. The beverage according to claim 1 or 2, wherein, Lactic acid bacteria are selected from one or more of the group consisting of bacteria of the genus Lactococcus.

4. A method for manufacturing a beverage, characterized in that, In the manufacture of beverages, the beverages contain one or more dead cells of one or more lactic acid bacteria at a concentration of more than 1.3 billion cells / L, and contain hesperidin at a concentration of 10 to 1000 mg / L.

5. A method for inhibiting off-flavors in a culture medium for lactic acid bacteria in a beverage, characterized in that, In the manufacture of beverages, the beverages contain one or more dead cells of one or more lactic acid bacteria at a concentration of more than 1.3 billion cells / L, and contain hesperidin at a concentration of 10 to 1000 mg / L.

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