JP7740906B2 - Fermentation production method - Google Patents

Description

NPMD NITE P-03235 NPMD NITE P-02661 IPOD FERM BP-10123

The present invention relates to a method for producing a fermented product, etc.

Ginseng (medicinal ginseng), including Panax ginseng (C.A. Meyer), has been used as a medicinal herb since ancient times, mainly in China, the Korean Peninsula, and Japan. Its useful components are said to be saponins (ginseng saponins) with a dalaman skeleton called ginsenosides, and research has revealed that they have various physiological effects, including antioxidant, anti-diabetic, anti-obesity, anti-hypertension, and anti-cancer properties (Non-Patent Document 1).

Ginseng saponins are known to contain various ginsenosides. Among these, protopanaxadiol, such as ginsenosides Rb1, Rb2, Rc, and Rd, is thought to be metabolically converted by intestinal bacteria to 20(S)-protopanaxadiol 20-O-β-d-glucopyranoside (also known as M1 or compound K), which then passes through the intestinal wall and enters the bloodstream, where it exerts the physiological effects described above.
Regarding M1, it has been reported that fermented ginseng contains M1, and that tests on mice and humans have shown that stress is reduced compared to before fermentation (Patent Document 1) and that the sleep-improving effect is increased (Non-Patent Document 2).

Fermented ginseng is obtained by fermenting ginseng with a microorganism, and Patent Document 1 discloses a fermentation method using a microorganism that produces at least one enzyme selected from the group consisting of β-glucosidase, α-arabinosidase, and α-rhamnosidase, and that can be added to foods, specifically, Lactobacillus casei Hasegawa strain (FERM BP-10123).

Patent No. 5204771

Journal of Ginseng Research 37, 261-268(2013) SLEEP 32, 413-421(2009)

The object of the present invention is to provide a novel method for producing fermented products.

Another object of the present invention is to provide a method for producing a fermented product that can efficiently produce M1.

Another object of the present invention is to provide a novel fermented product.

Another object of the present invention is to provide a novel microorganism.

After extensive research, the inventors discovered that a novel method for producing fermented products can be provided by combining enzyme treatment with fermentation treatment using Lactobacillus lactic acid bacteria (e.g., Lactobacillus casei Hasegawa strain), and that M1 can be efficiently produced by using a specific enzyme (i.e., lactase) in the enzyme treatment, leading to the completion of the present invention.

That is, the present invention relates to the following inventions.
[1]
A method for producing fermented carrots through enzymatic treatment with lactase (galactosidase) and fermentation treatment with Lactobacillus lactic acid bacteria (lactic acid bacteria belonging to the Lactobacillus genus).
[2]
The method according to [1], wherein the ginseng comprises at least one species selected from the group consisting of Panax ginseng, Panax notoginseng, American ginseng, Bamboo ginseng, Himalayan ginseng, and Vietnamese ginseng.
[3]
The method according to [1] or [2], wherein the enzyme treatment is carried out at an enzyme concentration of 0.01 to 20 w/v% at 30 to 60°C for 3 to 72 hours.
[4]
The method according to any one of [1] to [3], wherein the Lactobacillus lactic acid bacteria comprises at least one species selected from Lactobacillus casei and Lactobacillus brevis.
[5]
The method according to any one of [1] to [4], wherein the Lactobacillus lactic acid bacteria comprises at least one species selected from the group consisting of Lactobacillus casei Hasegawa strain (FERM BP-10123), Lactobacillus casei NBRC15883 strain, Lactobacillus brevis DNBL1871 strain (accession number NITE P-02661), and Lactobacillus brevis DNBL1889 strain (accession number NITE AP-03235, accession number NITE P-03235).
[6]
The method according to any one of [1] to [5], wherein the Lactobacillus lactic acid bacteria include Lactobacillus brevis DNBL1889 strain (accession number NITE AP-03235, accession number NITE P-03235).
[7]
The method according to any one of [1] to [6], wherein the enzyme treatment is carried out simultaneously with or before the fermentation treatment.
[8]
The method according to any one of [1] to [6], wherein the fermentation treatment is carried out simultaneously with or before the enzyme treatment.
[9]
The method according to any one of [1] to [8], wherein the fermented product contains M1.
[10]
A fermented carrot product obtained by the method according to any one of [1] to [9].
[11]
A fermented carrot product containing M1 at a ratio of 0.75% by mass or more (e.g., 0.8% by mass or more, 0.85% by mass or more, 0.9% by mass or more).
[12]
A carrot fermentation product containing M1, wherein the M3/M1 area ratio in HPLC is 0.4 or less.
[13]
The fermented product according to [11] or [12], which does not contain ginsenoside Rb1.
[14]
The fermented product according to [12] or [13], containing M1 at a rate of 0.4% by mass or more.
[15]
A composition containing the fermented product according to any one of [10] to [14].
[16]
Lactobacillus brevis strain DNBL1889 (accession number NITE AP-03235, accession number NITE P-03235).

According to the present invention, a novel method for producing a fermented product can be provided.
Such a method combines a specific enzyme treatment with a fermentation treatment using specific lactic acid bacteria, and can efficiently carry out fermentation (e.g., metabolism or hydrolysis) [e.g., can efficiently produce M1 (can produce a fermented product containing a high proportion of M1)].

In another aspect of the present invention, a novel fermented product can be provided. Such a fermented product can be produced, for example, by the above-mentioned method, i.e., a method that combines a specific enzyme treatment with a fermentation treatment using specific lactic acid bacteria, and can have characteristics such as a high content of M1.

In another aspect of the present invention, a novel microorganism is provided. Such a microorganism can be used in the fermentation process of carrots, and can be particularly suitably used as a lactic acid bacterium in the above-mentioned method (i.e., a method that combines a specific enzyme treatment with a fermentation process using a specific lactic acid bacterium).

[Manufacturing method]
In the present invention, a fermented carrot product is produced through an enzymatic treatment with lactase (galactosidase) and a fermentation treatment with Lactobacillus lactic acid bacteria (lactic acid bacteria belonging to the genus Lactobacillus).

As ginseng (medicinal ginseng), for example, Araliaceae ginseng (Araliaceae ginseng) can be used. Such ginseng may typically be capable of producing M1 through fermentation.

Representative examples of ginseng (Araliaceae ginseng) include Panax ginseng (Korean ginseng: Panax C.A. Meyer), Panax notoginseng Burk., American ginseng (Panax quinquefolium L.), bamboo-jointed ginseng (Panax japonicus C.A. Meyer), Himalayan ginseng (Panax pseudo-ginseng Qall. subsp. Himalaicus Hara), and Vietnamese ginseng (Panax Vuetnamensis Ha et Grussv.).

Carrots may be used alone or in combination of two or more types.

Carrots may be natural (unprocessed) or processed. Examples of processed products include dried carrots, cut carrots, crushed carrots, extracts, pastes, etc. Conventional methods can be used for drying, cutting, crushing, extraction, and pasting.

Carrots can be dried raw ginseng, white ginseng, red ginseng, etc.

The size of the carrots (processed products) is not particularly limited, but may be, for example, an average major axis of 0.2 mm or less.
The carrots may be commercially available.

There are no particular limitations on the part of the carrot that can be used, and any part can be used. Examples include the root, stem, leaf, flower bud, fruit, and whole plant. One or more of these can be used. Roots are preferred, and lateral roots and taproots are more preferred.

The enzyme used in enzymatic treatment is lactase [galactosidase (β-galactosidase)].

The enzymes may include lactase, but may also include other enzymes.

The enzyme is not particularly limited in terms of the microorganism from which it is derived. For example, lactase may be derived from a fungus (e.g., the genus Aspergillus [e.g., Aspergillus oryzae (Aspergillus oryzae)], etc.) or a yeast (e.g., the genus Kluveromyces [e.g., Kluveromyces lactis], etc.).

The enzyme may be a commercially available product. For example, lactase is available from Amano Enzyme Co., Ltd. as Lactase F "Amano."

The enzyme treatment method is not particularly limited, and is usually carried out by contacting the enzyme with the carrots.

The enzyme treatment may also be carried out in the presence of a medium (reaction medium) (e.g., in water, etc.).

In the enzymatic treatment, the amount (proportion) of the enzyme (lactase) used may be, for example, 0.001 part by mass or more, preferably 0.005 part by mass or more, and more preferably 0.01 part by mass or more, per 1 part by mass of carrots.

The amount (proportion) of the enzyme (lactase) used may be, for example, 0.2 parts by mass or less, preferably 0.1 parts by mass or less, and more preferably 0.08 parts by mass or less per 1 part by mass of carrot.

In the present invention, it is possible to efficiently obtain a fermented product even when using a small amount of enzyme (lactase).

In enzyme treatment (e.g., enzyme treatment in a medium such as water), the proportion of the enzyme (or concentration, e.g., the proportion relative to the medium such as water) may be, for example, 0.01 w/v% or more (e.g., 0.05 w/v% or more), preferably 0.1 w/v% or more (e.g., 0.5 w/v% or more), and more preferably 1 w/v% or more.
In the enzyme treatment, the upper limit of the enzyme proportion (concentration) may be, for example, 50 w/v%, 40 w/v%, 30 w/v%, 20 w/v%, 15 w/v%, 10 w/v%, 8 w/v%, 5 w/v%, etc.

In enzyme treatment (e.g., enzyme treatment in a medium such as water), the proportion of carrot (or concentration, e.g., the proportion relative to the medium such as water) may be, for example, 1 w/v% or more (e.g., 2 w/v% or more), preferably 5 w/v% or more (e.g., 10 w/v% or more), more preferably 15 w/v% or more, or may be 50 w/v% or less, 40 w/v% or less, 30 w/v% or less, etc.

The temperature (enzyme treatment temperature) during enzyme treatment may be, for example, 15 to 80°C (e.g., 20 to 70°C), preferably 30 to 60°C, and more preferably 40 to 55°C.

The time period for enzyme treatment (enzyme treatment time) may be, for example, 1 hour or more, preferably 3 hours or more, and more preferably 8 hours or more (e.g., 15 hours or more), or may be 10 days or less, 8 days or less, 5 days or less, 3 days (72 hours) or less, 48 hours or less, 24 hours or less, etc.

The fermentation process is carried out using (in the presence of) Lactobacillus lactic acid bacteria (lactic acid bacteria belonging to the Lactobacillus genus) as the microorganism.

Examples of Lactobacillus lactic acid bacteria include Lactobacillus acidophilus, Lactobacillus gasseri, Lactobacillus mali, Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus johnsonii, Lactobacillus gallinarum, and Lactobacillus casei. Examples include Bacillus amylovorus (L. amylovorus), Lactobacillus brevis (L. brevis), Lactobacillus rhamnosus (L. rhamnosus), Lactobacillus kefir (L. kefir), Lactobacillus paracasei (L. paracasei), Lactobacillus crispatus (L. crispatus), and Lactobacillus pentosus (L. pentosus).

There are no particular limitations on the strain of each bacterial species.

For example, examples of Lactobacillus casei (strains) include Lactobacillus casei Hasegawa strain (FERM BP-10123), Lactobacillus casei ATCC393 strain, and Lactobacillus casei NBRC15883 strain.

Furthermore, for example, Lactobacillus brevis (strains) that can be used include Lactobacillus brevis ATCC14869 strain, Lactobacillus brevis JCM1559 strain, Lactobacillus brevis NBRC12005 strain, Lactobacillus brevis DNBL1867 strain, Lactobacillus brevis DNBL1868 strain, Lactobacillus brevis DNBL1869 strain, Lactobacillus brevis DNBL1870 strain, Lactobacillus brevis DNBL1871 strain (accession number NITE P-02661), and Lactobacillus brevis DNBL1889 strain (accession number NITE AP-03235, accession number NITE P-03235).

The above microorganisms may be deposited. For example, Lactobacillus casei Hasegawa strain (FERM BP-10123) was deposited on August 11, 2003 (deposit date) at the National Institute of Advanced Industrial Science and Technology (AIST), Patent Organism Depositary (Address: Central 6, 1-1-1 Higashi, Tsukuba City, Ibaraki Prefecture, Japan, Postal Code 305-8566) under accession number FERM BP-10123. Lactobacillus brevis DNBL1871 strain (Accession Number NITE P-02661) was deposited at the National Institute of Technology and Evaluation (NPMD), Patent Microorganism Depositary (Address: 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Postal Code 292-0818). It was deposited at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD), Room 122, Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture, 292-0818, Japan, under accession number NITE P-02661 on March 6, 2018 (date of deposit), and the Lactobacillus brevis strain DNBL1889 (accession number NITE AP-03235, accession number NITE P-03235) was received at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary (NPMD), Room 122, Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture, 292-0818, Japan, under accession number NITE AP-03235 on June 22, 2020 (date of receipt).

Among these Lactobacillus lactic acid bacteria, Lactobacillus casei (for example, Lactobacillus casei Hasegawa strain) and Lactobacillus brevis (for example, Lactobacillus brevis DNBL1889 strain, Lactobacillus brevis DNBL1871 strain) may be preferably used.
In particular, Lactobacillus brevis strain DNBL1889, Lactobacillus brevis strain DNBL1871, etc. can dramatically improve the efficiency of fermentation (metabolism or hydrolysis) by combining them with an enzyme treatment (and further by combining them with other Lactobacillus lactic acid bacteria (e.g., Lactobacillus casei Hasegawa strain)).

The Lactobacillus brevis strain DNBL1889 is a novel microorganism (lactic acid bacterium). Therefore, the present invention encompasses this novel microorganism. As mentioned above, this novel microorganism is useful in combination with enzyme treatment, and also appears to have properties that make it useful for the fermentation (metabolism or hydrolysis) of ginsenosides, particularly ginsenoside F2.

Lactobacillus lactic acid bacteria (species, strains) may be used alone or in combination of two or more types.

In addition, as long as the microorganisms (fermentation process) contain (use) Lactobacillus lactic acid bacteria, other microorganisms may also be included (used).

Fermentation is typically carried out by bringing carrots into contact with microorganisms (Lactobacillus lactic acid bacteria).

The fermentation process (fermentation method) may be carried out in a medium (in a medium).
For example, fermentation may be carried out in (via) a medium.
When using such a medium, for example, fermentation can be carried out by inoculating a medium (culture medium) containing carrots with microorganisms.

The medium may be sterilized (treated) before being inoculated with the microorganism. Sterilization methods are not particularly limited, and examples include heat sterilization, high-pressure steam sterilization, and filtration sterilization.

The medium (culture medium) is not particularly limited, but may be, for example, a medium containing a carbon source, nitrogen source, mineral source, etc. commonly used in the cultivation of microorganisms, and may be either a natural medium or a synthetic medium. Preferably, a liquid medium may be used.

The nitrogen source is not particularly limited, and examples of inorganic nitrogen sources include ammonia and ammonium salts, while examples of organic nitrogen sources include peptone, polypeptone, urea, amino acids, proteins, and peptides such as soybean peptides. The nitrogen source is preferably peptone, polypeptone, or peptide. Furthermore, examples of mineral sources are not particularly limited, and include yeast extract, meat extract, and, in addition, potassium monohydrogen phosphate, magnesium sulfate, and the like, which contain K, P, Mg, S, and the like. These nitrogen and mineral sources can be used alone or in combination of two or more.
The concentration of the nitrogen source in the medium (culture medium) is not particularly limited as long as it is a normal concentration at which the microorganism can grow. The concentration of the nitrogen source at the start of culture is usually preferably about 0.05 to 10% by weight, more preferably about 0.1 to 5% by weight.

In addition to the nitrogen and mineral sources, the medium (culture medium) may further contain a carbon source, inorganic substances, pH buffers, etc. Examples of inorganic substances include, but are not limited to, ammonium sulfate, potassium phosphate, magnesium chloride, salt, iron, manganese, molybdenum, and various vitamins, which can be used alone or in combination of two or more. Examples of pH buffers include, but are not limited to, calcium carbonate, etc.

In fermentation treatment (e.g., fermentation treatment in a medium (culture medium)), the proportion of carrots (or concentration, e.g., the proportion relative to the medium (fermentation liquid)) is not particularly limited and can be selected appropriately depending on the type, shape, dryness, culture conditions, etc., but may be selected from a range of, for example, approximately 0.1 w/v% or more, 1 w/v% or more (e.g., 2 w/v% or more), preferably 5 w/v% or more (e.g., 10 w/v% or more), more preferably 15 w/v% or more, or may be 50 w/v% or less, 40 w/v% or less, 30 w/v% or less, etc.

Furthermore, in the fermentation treatment, the proportion of carrots may be, for example, 0.1 to 100 parts by mass (e.g., 0.5 to 80 parts by mass), preferably 1 to 50 parts by mass (e.g., 2 to 30 parts by mass), and more preferably 3 to 20 parts by mass (e.g., 5 to 18 parts by mass, 10 to 15 parts by mass, etc.) per 100 parts by mass of medium (medium not containing carrots, total amount of medium excluding carrots).

The carrots may be dried (for example, dried for a specified period of time (approximately 1 to 6 hours) at an internal temperature of approximately 100 to 180°C), and if they are not dried, the amount (ratio) used may be converted to the amount of dried carrots (for example, if undried carrots are used, the amount may be converted to the amount of dried carrots used).

During fermentation, the pH of the system (or medium (culture medium)) may be, for example, approximately 3 to 7, and preferably approximately 5 to 6.5. The pH may be controlled or adjusted using an acid or alkali.

During fermentation, the temperature (fermentation temperature) may be approximately 5°C or higher (e.g., 8°C or higher), for example, 10°C or higher (e.g., 12-60°C), preferably 15°C or higher (e.g., 18-55°C), more preferably 20°C or higher (e.g., 22-50°C), or may be 25-40°C (e.g., 25-37°C, 28-33°C), etc.

The time (fermentation treatment time) for fermentation can be selected appropriately depending on the fermentation conditions [e.g., the composition of the system (e.g., the amount of microorganisms), temperature, etc.], but may be, for example, 6 hours or more (e.g., 12 hours or more, 1 day or more), 2 days or more, 3 days or more, 5 days or more, 7 days or more, etc. The upper limit of the fermentation time is not particularly limited, and may be 3 months, 2 months, 1 month, 25 days, 21 days, 14 days, 10 days, etc.

As described above, the present invention combines enzyme treatment and fermentation treatment, but these treatments may be carried out simultaneously (or in parallel), or one treatment may be carried out after the other.

The other treatment may be carried out after separating the processed product from the other treatment, or after the other treatment, it may be carried out in the same system without separation.

In particular, in the present invention, it is preferred that the enzyme treatment be carried out simultaneously with or before the fermentation treatment, and it is particularly preferred that the enzyme treatment be carried out before (prior to) the fermentation treatment.
By combining the two treatments in this way, it becomes easier to obtain a fermented product (for example, a fermented product containing a high content of M1) more efficiently.
On the other hand, in the present invention, from the viewpoints of easily reducing thermal history, easily reducing damage to materials, and improving the efficiency of the manufacturing process, it is preferable to carry out the fermentation treatment simultaneously with or before the enzyme treatment, and in particular, before (prior to) the fermentation treatment.

The method of the present invention may include (or undergo) an enzyme treatment (enzyme treatment step) and a fermentation treatment (fermentation treatment step), and may also include (or undergo) other treatments (steps) as necessary. For example, before, during, and/or after these treatments, treatments such as filtration, centrifugation, concentration, ultrafiltration, lyophilization, powderization, fractionation, etc. may be carried out as desired.
As a specific example, sterilization (heat treatment, etc.) may be carried out after the fermentation treatment, which can kill the microorganisms used in the fermentation treatment.
It should be noted that such sterilization treatment may be omitted (or may be replaced by a step after the fermentation step) depending on the step after the fermentation treatment.
For example, when enzyme treatment is carried out in parallel with or after fermentation treatment, the enzyme treatment may be carried out without sterilization treatment (heat treatment, etc.).

[Fermented products, compositions, and their uses]
In this manner, a fermented product (fermented carrot product) is obtained.
Such fermentations typically contain M1 [20(S)-protopanaxadiol 20-O-β-d-glucopyranoside, compound K].

In such fermented products, M1 is often produced efficiently through the above-mentioned method. For example, when the amount of M1 produced (content in the fermented product) is taken as 1 when only fermentation treatment [fermentation treatment using Lactobacillus lactic acid bacteria (e.g., Lactobacillus casei Hasegawa strain (FERM BP-10123)] is performed, the amount can be made greater than 1, preferably 1.1 or more, more preferably 1.2 or more, and particularly 1.3 or more (e.g., 1.4 or more, 1.5 or more, 1.6 or more, 1.7 or more, 1.8 or more, 2 or more), etc.

The content of M1 in the fermented product (content in the fermented product) is not particularly limited, but may be, for example, 0.1% by mass or more (e.g., 0.2% by mass or more), preferably 0.3% by mass or more (e.g., 0.35% by mass or more), more preferably 0.4% by mass or more (e.g., 0.45% by mass or more), or 0.5% by mass or more (e.g., 0.6% by mass or more, 0.7% by mass or more, 0.75% by mass or more, 0.8% by mass or more, 0.9% by mass or more, 1% by mass or more, 1.1% by mass or more, 1.2% by mass or more, 1.3% by mass or more, 1.4% by mass or more), etc.

The fermented product may also contain components other than M1 (e.g., ginsenoside Rb2, ginsenoside Rc, ginsenoside Rd, ginsenoside Re, ginsenoside Rg1, gypenoside XVII, M2 [20(S)-protopanaxadiol 20-O-[β-L-arabinopyranosyl(1→6)-β-D-glucopyranoside], M3 [20(S)-protopanaxadiol 20-O-[β-L-arabinofuranosyl(1→6)-β-D-glucopyranoside], etc.]).

In the fermented product, the ratio of M3 to M1 (M3/M1), expressed as an HPLC area ratio (peak area ratio), can be selected from a range of approximately 0.5 or less (e.g., 0.48 or less), and may be 0.45 or less (e.g., 0.43 or less), preferably 0.42 or less (e.g., 0.4 or less, 0.38 or less, 0.35 or less, 0.32 or less, 0.3 or less, 0.28 or less), more preferably 0.25 or less (e.g., 0.24 or less, 0.22 or less, 0.21 or less), or 0.2 or less (e.g., 0.19 or less, 0.18 or less, 0.16 or less, 0.15 or less, etc.).

The fermentate may not contain M3 (or may contain M1, with the M3/M1 area ratio in HPLC being 0).

The fermented product may also contain ginsenoside Rb1, but in many cases the amount of ginsenoside Rb1 is low, and in particular it may not contain ginsenoside Rb1 (or it may contain M1, and the ginsenoside Rb1/M1 value in terms of area ratio in HPLC may be 0).

The present invention makes it easy to obtain fermented products with relatively low levels of M3 and ginsenoside Rb1.

The presence or absence and proportion of each component in such a fermented product may be confirmed, for example, by HPLC (HPLC analysis).
Furthermore, "not containing" may mean, for example, that it is not detected (no peak is detected) in HPLC (it is at the detection limit) (the same applies hereinafter).

The present invention also includes such fermented products. Note that these fermented products may or may not have been obtained by the above-mentioned method (they may be obtained by a different method).

The fermented product of the present invention may be separated as is, or a portion thereof (e.g., a fraction containing M1) may be separated. The fermented product may also contain components used in the above-mentioned methods (e.g., enzymes, lactic acid bacteria, medium components, etc.).

The fermented product of the present invention may constitute a composition. Therefore, the present invention also includes compositions containing the fermented product.

The fermented product (or composition) of the present invention can be used for various purposes, such as the prevention, amelioration, and/or treatment of various diseases or symptoms. Specifically, the fermented product (or composition) of the present invention may be used for, for example, anti-tumor, anti-stress, diabetes prevention (amelioration, treatment), anti-obesity, hair growth, anti-amnesia, hepatoprotective effect, anti-arteriosclerosis, anti-aging, anti-hyperlipidemic, anti-thrombotic, blood pressure lowering effect, immune function improvement, blood sugar lowering effect, analgesic effect, cardiotonic effect, anti-inflammatory effect, peripheral blood flow improvement effect, hemostatic effect, antioxidant effect, anti-muscle atrophy effect, bone mass increase effect, anti-infectious disease (anti-influenza, etc.), neuroprotective effect, anti-fatigue, skin beautification, whitening effect, etc.

The fermented product (or composition) of the present invention may be contained in (compounded into) a food or drink, or may be in any suitable form. For example, the fermented product (or composition) of the present invention may be formulated into oral or parenteral preparations such as tablets, powders, granules, capsules, liquids, emulsions, elixirs, suspensions, syrups, lozenges, inhalants, suppositories, injections, ointments, eye ointments, eye drops, nasal drops, ear drops, poultices, and lotions.

When ingesting (administering) the fermented product (or composition) of the present invention, the conditions for ingestion (administration) can be selected appropriately depending on the form, purpose of administration, type, age, weight, and symptoms of the recipient. For example, the effective human dose of M1 may be set to approximately 0.01 to 100 mg/kg (e.g., 1 to 50 mg/kg) per day. Administration may be performed once or in several divided doses within the desired dosage range within a day. The administration period is also optional.

The subjects to be inoculated (administered) with the fermented product (or composition) of the present invention may be humans or non-human animals (e.g., mammals such as dogs, cats, rabbits, cows, horses, goats, monkeys, and mice). Non-human animals may also be pets, livestock, or laboratory animals (e.g., mice, rats, guinea pigs, monkeys, etc.).

The fermented product (or composition) of the present invention may contain other components such as carriers, bases, and additives depending on the intended use and form (e.g., use form, formulation form), etc. Specifically, it may contain, for example, excipients, binders, lubricants, colorants, flavorings, and, if necessary, stabilizers, emulsifiers, absorption enhancers, surfactants, pH adjusters, preservatives, antioxidants, etc.

The content of the fermented product in the composition can be selected appropriately depending on the intended use, form, etc., but may be, for example, 0.01% by mass or more (e.g., 0.05% by mass or more, 0.1% by mass or more, 0.3% by mass or more, 0.5% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 10% by mass or more, etc.), or less than 100% by mass (e.g., 99% by mass or less, 95% by mass or less, 90% by mass or less, 80% by mass or less, 50% by mass or less, 30% by mass or less, 20% by mass or less, 10% by mass or less, 5% by mass or less, 3% by mass or less, 1% by mass or less, etc.).

The present invention will be explained in detail below using examples, but the present invention is not limited to these examples.

The lactic acid bacteria and enzymes used in the examples are as follows:
Lactobacillus casei Hasegawa strain (accession number: FERM BP-10123, hereinafter referred to as A221 strain)
Lactobacillus casei NBRC15883 strain (also known as JCM1134T, hereinafter referred to as 15883 strain)
Lactobacillus brevis strain DNBL1889 (accession number NITE AP-03235, accession number NITE P-03235, hereinafter referred to as strain DNBL1889)
Lactobacillus brevis strain DNBL1871 (accession number NITE P-02661, hereinafter referred to as strain DNBL1871)

Lactase: Lactase F (manufacturer: Amano Enzyme Co., Ltd.)
Cellulase: Y-NC (manufacturer: Yakult Kogyo Co., Ltd.)
β-glucosidase: Aromase (manufacturer: Amano Enzyme Co., Ltd.)

Test Example 1
(1) Each lactic acid bacterium was inoculated into 6 mL of a general lactic acid bacterium inoculation medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and pre-cultured overnight at 28°C.
(2) 6.5 g of ginseng powder was added to 40 mL of distilled water and heated at 105°C for 5 minutes.
(3) After cooling to 50°C or below, 400 mg of each enzyme was added, and the mixture was shaken at 55°C and 100 rpm to carry out the enzyme reaction for 17 hours (when only the enzyme treatment was carried out, i.e., when only the lactic acid bacteria fermentation treatment was carried out, this step (3) (and further heating at 105°C for 5 minutes in step (2)) was not carried out).
(4) After the reaction was completed, 0.5 g of yeast extract, 0.25 g of soybean peptone, and 0.5 g of calcium carbonate were added and mixed, and this was used as a fermentation medium and heat sterilized at 121°C for 15 minutes (only when lactic acid bacteria treatment was performed, i.e., when only enzyme treatment was performed, this step (4) and the step (5) described below were not performed).
(5) 1.5 mL of each lactic acid bacteria preculture solution was added to the fermentation medium, and the mixture was allowed to stand at 28°C for static cultivation.
(6) After 6 days, the samples were heated (at 110°C for 20 minutes) to kill the lactic acid bacteria. The fermentation medium was thoroughly shaken and 0.8 g of each sample was collected. The same amount of ethanol was added to each sample, and the mixture was stirred and centrifuged (at 10,000 rpm for 5 minutes) to obtain a supernatant. One mL of the supernatant was loaded onto a Sep-Pak C18 cartridge (manufactured by Waters) for adsorption. The cartridge was washed with distilled water and a 40% aqueous methanol solution, and then eluted with methanol. (When only enzyme treatment was performed, i.e., when lactic acid bacteria fermentation treatment was not performed, the enzyme-treated solution obtained in step (3) was used instead of the fermentation medium.)
(7) 10 μL of the eluted methanol solution was taken and analyzed for M1, M3, and Rb1 by high performance liquid chromatography (HPLC) under the conditions below, and the relative value to the peak area of M1 when no enzyme treatment was performed and only lactic acid bacteria fermentation treatment using the A221 strain was performed (the value at the beginning of the table below, i.e., when the peak area of M1 when the enzyme used in step (3) was "not used" and the lactic acid bacteria used in step (5) was "A221" is set to 1), the M3/M1 ratio (only when M1 was detected), and the ginsenoside Rb1/M1 ratio (only when M1 was detected) were quantified.

<HPLC analysis condition 1 (analysis of M1 and M3)>
Column: YMC-Pack ODS-A (250 x 4.6 mm)
Analysis temperature: 40℃
Eluent: 60% (V/V) acetonitrile Flow rate: 1 mL/min
Detection: 203 nm

<HPLC analysis condition 2 (analysis of Rb1 and M1)>
Column: YMC-Pack ODS-A (250 x 4.6 mm)
Analysis temperature: 40℃
Eluent:
0-5 min: 35% (V/V) acetonitrile 5-35 min: 35% (V/V) → 60% (V/V) acetonitrile gradient 35-50 min: 60% (V/V) acetonitrile Flow rate: 1 mL/min
Detection: 203 nm

The results are shown in the table below.

Test Example 2
In "(7)" of Test Example 1, each step was carried out in the same manner as in Test Example 1, except that the relative value to the peak area of M1 when only lactic acid bacteria fermentation treatment with the A221 strain was carried out without enzyme treatment was replaced with the relative value to the peak area of M1 when only lactic acid bacteria fermentation treatment with the A221 strain and the NBRC15883 strain was carried out without enzyme treatment (the value at the beginning of the table below, i.e., when the peak area of M1 when "no enzyme" was used in step (3) and the lactic acid bacteria "A221 and NBRC15883" were used in step (5) was set to 1).

The results are shown in the table below.

Test Example 3
(1) Each lactic acid bacterium was inoculated into 6 mL of a general lactic acid bacterium inoculation medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and pre-cultured overnight at 28°C.
(2) 13.0 g of Panax ginseng (Higashiginseng) powder was added to 40 mL of distilled water and heated at 105°C for 5 minutes.
(3) After cooling to 50°C or below, 400 mg of enzyme was added, and the mixture was shaken at 55°C and 100 rpm to carry out an enzyme reaction for 17 hours (when only enzyme treatment was carried out, i.e., when only lactic acid bacteria fermentation treatment was carried out, this step (3) (and further heating at 105°C for 5 minutes in step (2)) was not carried out).
(4) After the reaction was completed, 1.0 g of yeast extract, 0.5 g of soybean peptone, and 1.0 g of calcium carbonate were added and mixed, and the mixture was used as a fermentation medium and sterilized by heating at 121° C. for 15 minutes.
(5) 3.0 mL of each lactic acid bacteria preculture solution was added to the fermentation medium, and the mixture was allowed to stand at 28°C for static cultivation.
(6) After 10 days, the fermentation medium was sterilized by heating at 121°C for 15 minutes and then cooled to room temperature.
(7) The fermentation medium of (6) was frozen in a freezer at −20° C., and then freeze-dried for 5 days to obtain a powder.
(8) 20 mg of the obtained powder was weighed out, 400 μL of 50% aqueous ethanol solution was added, and the mixture was stirred and then centrifuged (10,000 rpm for 5 minutes). 10 μL of the supernatant was analyzed under the HPLC analysis condition 1 described in Test Example 1, and the M1 content [W/W (%) (mass %)] in the powder was determined.
In addition, in the same manner as in Test Example 1, M1, M3, and Rb1 were analyzed, and the M3/M1 ratio (only those in which M1 could be detected) and the ginsenoside Rb1/M1 ratio (only those in which M1 could be detected) were quantified.

The results are shown in the table below.

Test Example 4
(1) Each lactic acid bacterium was inoculated into 6 mL of a general lactic acid bacterium inoculation medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and pre-cultured overnight at 28°C.
(2) Separately, 6.5 g of ginseng powder was added to 40 mL of distilled water, and then 0.5 g of yeast extract, 0.25 g of soybean peptone, and 0.5 g of calcium carbonate were added and mixed. This was used as a fermentation medium and sterilized by heating at 121°C for 15 minutes.
(3) 1.5 mL of each lactic acid bacteria preculture solution was added to the fermentation medium, and the mixture was allowed to stand at 28°C for static cultivation.
(4) After 6 days, 400 mg of each enzyme was added to the fermentation medium, and the mixture was shaken at 55°C and 1000 rpm to carry out the enzyme reaction for 17 hours (when only the enzyme treatment was carried out, i.e., when only the lactic acid bacteria fermentation treatment was carried out, this step (4) and the step (5) described below were not carried out).
(5) After the enzyme reaction was completed, the mixture was heated at 90°C for 15 minutes to inactivate the enzyme.
(6) After shaking well, 0.8 g of each sample was collected, to which the same amount of ethanol was added, and after stirring, the mixture was centrifuged (10,000 rpm, 5 minutes) to obtain a supernatant. 1 mL of this supernatant was added to a Sep-Pak C18 cartridge (manufactured by Waters) for adsorption, washed with distilled water and 40% aqueous methanol solution, and then eluted with methanol.
(7) 10 μL of the eluted methanol solution was taken and analyzed for M1, M3, and Rb1 by high performance liquid chromatography (HPLC) under the same conditions as in Test Example 1. The relative value to the peak area of M1 when no enzyme treatment was performed and only lactic acid bacteria fermentation treatment using the A221 strain was performed, the M3/M1 ratio (only when M1 was detected), and the ginsenoside Rb1/M1 ratio (only when M1 was detected) were quantified.

The results are shown in the table below.

The present invention provides a method for producing fermented carrots.

Claims (9)

A method for producing a fermented carrot product through an enzymatic treatment with lactase and a fermentation treatment with Lactobacillus lactic acid bacteria ,
The method, wherein the Lactobacillus lactic acid bacteria include Lactobacillus brevis DNBL1889 strain (Accession No. NITE AP-03235, Accession No. NITE P-03235) . The method of claim 1, wherein the ginseng comprises at least one species selected from the group consisting of Panax ginseng, Panax notoginseng, American ginseng, Bamboo ginseng, Himalayan ginseng, and Vietnamese ginseng. The method of claim 1 or 2, wherein the enzyme treatment is carried out at an enzyme concentration of 0.01 to 20 w/v% at 30 to 60°C for 3 to 72 hours. The method according to any one of claims 1 to 3, wherein the Lactobacillus lactic acid bacteria further comprise at least one species selected from Lactobacillus casei and Lactobacillus brevis (excluding Lactobacillus brevis DNBL1889 strain (Accession No. NITE AP-03235, Accession No. NITE P-03235)) . The method according to any one of claims 1 to 4, wherein the Lactobacillus lactic acid bacteria further comprises at least one selected from the group consisting of Lactobacillus casei Hasegawa strain (FERM BP-10123), Lactobacillus casei NBRC15883 strain, and Lactobacillus brevis DNBL1871 strain (Accession No. NITE P-02661 ) . The method according to any one of claims 1 to 5 , wherein the enzyme treatment is carried out simultaneously with or before the fermentation treatment. The method according to any one of claims 1 to 5 , wherein the fermentation treatment is carried out simultaneously with or before the enzyme treatment. The method according to any one of claims 1 to 7 , wherein the fermentation product contains 20(S)-protopanaxadiol 20-O-β-d-glucopyranoside . Lactobacillus brevis strain DNBL1889 (accession number NITE AP-03235, accession number NITE P-03235).