JP2013223491A - Method for enhancing capability to produce ethanol of starter for fermented milk product - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve the proliferative activity of L. fermentum in a preculture medium and enhance the capability to produce ethanol in a fermented milk product.SOLUTION: A method for enhancing the capability to produce ethanol includes: using, as a preculture medium of a starter for fermented milk product, a milk raw material medium to which a nitrogen source and/or divalent metal ions are added to culture L. fermentum.

Description

The present invention relates to a method for enhancing ethanol production ability of a starter for fermented dairy products, and more particularly to a method for enhancing ethanol production ability of Lactobacillus fermentum. Specifically, as a pre-culture medium for a starter for fermented dairy products, a milk raw material medium to which a nitrogen source and / or a divalent metal ion is added is used.
The present invention relates to a method for enhancing ethanol production ability for culturing fermentum.

  Microorganisms such as lactic acid bacteria are used in fermented foods and fermented dairy products, and are important as factors that determine the flavor and texture of the fermented foods and fermented dairy products. It is important as an element that helps to maintain and improve health by acting in the intestines of the body.

  Fermented dairy products (natural cheese, yogurt (fermented milk), lactic acid bacteria beverages, fermented butter, etc.) are produced by lactic acid fermentation. Usually, lactic acid bacteria are selected according to their use, and a starter (starter for fermented dairy products) in advance. Will also be prepared). That is, in general, various lactic acid bacteria are inoculated into each preculture medium and cultured to prepare a starter for fermented milk products. And the fermented milk product starter is added to each raw material for fermented milk products and fermented, and the fermented milk products are manufactured. At this time, skim milk, skim concentrated milk having a solid content concentration of about 10%, reduced skim milk, or the like is used as the milk raw material medium for the preculture medium. On the other hand, GYP medium, MRS medium, etc. are used for this preculture medium as a synthetic medium mainly for the purpose of studying lactic acid bacteria.

  In recent years, with the diversification of fermented dairy products, microorganisms that are active in the intestines of the human body and that are characterized by characteristics such as bacterial species that are beneficial for maintaining and promoting health and bacterial species that are effective for improving flavor As a target (target), a method of screening (selecting) and utilizing the microorganism for production of fermented milk products is adopted.

  Synthetic media are often used when selecting microorganisms due to the originally intended properties. However, in the synthetic medium, since the compounding components are expensive, the production cost of the fermented dairy product is increased, so that it is not used for the actual production of the fermented dairy product. At this time, in the preparation of the starter in the synthetic medium, even if the microorganisms actually selected have good growth properties, the nutrient requirements and growth environment conditions of the microorganisms may be limited. On the other hand, when preparing a starter, the milk raw material medium may deteriorate the growth properties of the actually selected microorganisms and may not fully exhibit the originally intended characteristics.

  For example, in Patent Document 1, in lactic acid bacteria having citrate fermentation ability such as Streptococcus lactis subsp. Diacetylactis and Leuconostoc cremoris, one or two of iron, copper and molybdenum are used as a culture substrate containing citric acid. It is disclosed that by adding more than seeds as inorganic salts or organic salts, the growth of the lactic acid bacteria is enhanced and the aromatic substance (diacetyl) produced from the lactic acid bacteria can be enhanced. In addition, for example, in Patent Document 2, by adding calcium carbonate, calcium phosphate, and soluble magnesium salt to a medium using milk (milk component) as a nitrogen source and a carbon source, while suppressing a decrease in pH associated with lactic acid fermentation, It is disclosed that the growth of lactic acid bacteria could be promoted.

By the way, for the purpose of increasing the ethanol content in natural cheese and enhancing the fruity scent of pineapple-like, the present inventors, as a lactic acid bacterium having high ethanol production ability, L.
Fermentum OLL203697 (Accession Number: FERM BP-11433) and L. fermentum OLL203731 (Accession Number: FERM BP-11434) were found and patent applications were filed (Japanese Patent Application No. 2011-237762). It was also found that if the ethanol content in natural cheese is 0.01 w / w% or more, the amount of ester produced by the action of microorganisms increases during ripening and the like, and the fruity aroma like pineapple can be enhanced. In order to produce natural cheese using these lactic acid bacteria as a starter, it was cultured in a preculture medium (starter medium), and natural cheese was produced. As a preculture medium, cells cultured in an MRS medium and a preculture medium For cells cultured in a reduced skim milk medium (solid content concentration: about 10% by weight), the inoculated L.
Although there was no difference in the number of fermentum bacteria, there was a problem that there was a difference in ethanol production in natural cheese.

JP-A-63-202390 Japanese Patent Laid-Open No. 8-116872

  An object of the present invention is to improve the productivity of L. fermentum in the preculture medium and enhance the ethanol-producing ability of L. fermentum in the fermented milk product.

As a result of intensive studies to solve the above-mentioned problems, the present inventors cultured L. fermentum as a starter for fermented dairy products, as a preculture medium, a nitrogen source and / or a milk raw material medium. It has been found that by adding divalent metal ions and culturing, not only the number of bacteria in the milk raw material medium increases, but also the amount of ethanol produced in natural cheese increases.
That is, the present invention includes the following.
[1] L. fermentum is cultured using a milk raw material medium to which a nitrogen source and / or a divalent metal ion is added as a pre-culture medium for a starter for fermented dairy products, And / or L.
A method for enhancing the ethanol production ability of fermentum.
[2] The method for enhancing ethanol production capability according to [1], wherein the milk raw material medium comprises skim milk having a solid content concentration of 8 to 12%, skim concentrated milk, and reduced skim milk.
[3] The method for enhancing ethanol production ability according to [1] above, wherein the nitrogen source is a yeast extract and / or a peptide derived from milk.
[4] The divalent metal ion is at least one or more of Fe ²⁺ , Cu ²⁺ , Zn ²⁺ , Ca ²⁺ , Mg ²⁺ and Mn ²⁺ , The method for enhancing ethanol production ability according to [1].
[5] A starter for fermented dairy products containing L. fermentum whose ethanol production ability is enhanced by the method for enhancing ethanol production ability of [1] to [4].
[6] A method for producing a fermented dairy product with an increased ethanol content, wherein the raw milk is inoculated with the starter according to [5] and cultured, fermented and / or aged.
[7] A fermented dairy product having a high ethanol content produced by the method for producing a fermented dairy product according to [6].

  According to the present invention, since the amount of ethanol produced by L. fermentum in fermented dairy products is increased, fermented dairy products having a high ethanol content including natural cheese can be provided. And as a result, fermented milk products which strengthened fruity fragrance like pineapple like natural cheese can be provided.

It is a graph which shows the number of bacteria of L. fermentum in the starter of L. fermentumOLL203697 (accession number FERM BP-11433) cultured in SM medium added with a combination of yeast extract and manganese yeast. The numerical values on the horizontal axis in the graph indicate starters cultured in SM medium to which yeast extract was added to 0, 0.05, 0.1, 0.25, 0.5 wt% and manganese yeast was added to a concentration of 0.2 wt%. Means. It is a graph which shows the number of bacteria of L. fermentum in the starter of L. fermentumOLL203697 (accession number FERM BP-11433) cultured in SM medium added with a combination of yeast extract and manganese yeast. The numerical value on the horizontal axis in the graph means a starter cultured in an SM medium to which manganese yeast is added to a concentration of 0.05, 0.1, and 0.2% by weight and yeast extract is added to a concentration of 0.25% by weight.

  Hereinafter, the present invention will be described in detail, but the present invention is not limited to the individual forms described below.

  In the method for enhancing ethanol production ability of the present invention, L. fermentum is used as a starter for fermented milk products, and a milk raw material medium is used as a pre-culture medium for the starter for fermented milk products. In the present invention, the “milk raw material medium” may be any medium that uses animal milk such as cow, sheep, goat, etc. as raw material, but non-fat milk, non-fat concentrated milk, reduced skim milk, etc. A medium containing the main component, a medium containing them alone, or a medium containing them in combination can be desirably used. At this time, in a medium comprising skim milk, skim concentrated milk, and reduced skim milk, the solid content concentration is preferably 8 to 12% by weight, more preferably 9 to 12% by weight, and further preferably 9 to 11% by weight.

In the method for enhancing ethanol production ability of the present invention, L. fermentum is used as a starter for fermented milk products, and a milk raw material medium to which nitrogen source is added (formulated) is used as a pre-culture medium for the starter. To do. Here, for the purpose of strengthening the nitrogen source in the milk raw material medium, proteins, peptides, amino acids, non-protein nitrogen and the like can be used, but yeast extracts and / or peptides derived from milk can be desirably used. At this time, as a nitrogen source, any yeast extract can be used as long as it can be obtained for foods and pharmaceuticals. For example, yeast extract Kyowa C (trademark), yeast extract Kyowa H (trademark) Yeast extract Kyowa L (trademark), Yeast extract Kyowa W (trademark), Yeast extract Takeda HR (trademark), Yeast extract Takeda SL-W (trademark), Yeast extract JT-21A (trademark), etc. can be used. Here, as a yeast extract to be added (mixed) to the milk raw material medium, L.
There is no particular limitation as long as an effective amount or concentration that can enhance the ethanol-producing ability of fermentum is used, and it can be used in combination with other nitrogen sources such as amino acids. However, L.
The concentration of yeast extract to be added (enhanced) to this milk raw material medium from the viewpoint of effectively enhancing the ethanol production ability of fermentum, from the viewpoint of preventing or suppressing the adverse effects of flavor, and from the viewpoint of reducing production costs Is preferably 0.05 wt% or more and 1 wt% or less, more preferably 0.1 wt% or more and 0.8 wt% or less, further preferably 0.2 wt% or more and 0.7 wt% or less, and particularly preferably 0.25 wt% or more and 0.6 wt% or less.

In addition, peptides derived from milk can be used as a nitrogen source. Enzymatic degradation whey and enzymes obtained by enzymatic degradation (hydrolysis) of whey, whey protein concentrate (WPC), and whey protein isolate (WPI) Degraded WPC, enzymatic degradation WPI, whey protein degradation product obtained by enzymatic degradation (hydrolysis) of whey protein, casein degradation product obtained by enzymatic degradation (hydrolysis) of casein, etc. It can be used with seeds or two or more. In the present invention, the “milk-derived peptide” may be any peptide derived from animal milk such as cow, sheep or goat, and plant milk such as soy milk. Here, as an amount to be added (mixed) to the milk raw material medium that is a pre-culture medium, L.
If it uses the effective amount and effective concentration which can enhance the ethanol production ability of fermentum, it will not specifically limit, It can also use together with other nitrogen sources, such as a yeast extract. For example, in the case of adding a casein hydrolyzate (mixture of amino acid and peptide), the concentration is preferably 0.05% by weight to 1% by weight, more preferably 0.1% by weight to 0.8% by weight, and more preferably 0.2% by weight. The content is more preferably 0.7% by weight or less and particularly preferably 0.25% by weight or more and 0.6% by weight or less. As another nitrogen source, for example, an amino acid may be used. Amino acids include alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, etc. These can also be used, and at least one or more of these can be used.

In the method for enhancing ethanol production ability of the present invention, milk using L. fermentum as a starter for fermented dairy products and adding (compounding) divalent metal ions as a pre-culture medium for the starter for fermented dairy products A raw material medium is used. At this time, as a divalent metal ion, any of inorganic salts and organic salts can be used as long as they can be obtained for foods or pharmaceuticals, and these can be used in at least one kind or two or more kinds. . Here, as a divalent metal ion to be added (strengthened) to the milk raw material medium, L.
Fe ²⁺ , Cu ²⁺ , Zn ²⁺ , Ca ² from the viewpoint of effectively enhancing the ethanol production ability of fermentum, from the viewpoint of preventing and suppressing the adverse effects of flavor, and from the viewpoint of reducing manufacturing costs ⁺ , Mg ²⁺ , Mn ^2+
Are preferred, Ca ²⁺ , Mg ²⁺ , Mn ²⁺ are more preferred, Mg ²⁺ , Mn ²⁺
Is more preferable, and Mn ²⁺ is particularly preferable. Therefore, it is desirable to use, for example, iron yeast, copper yeast, zinc yeast, magnesium sulfate, magnesium yeast, manganese yeast and the like, and these may be used alone or in combination. And from the same viewpoint as the above viewpoint, the total concentration of divalent metal ions to be added (strengthened) to the milk raw material medium as the preculture medium (when using two or more divalent metal ions, The total concentration of these divalent metal ions) is preferably 0.03 mM or more and 2 mM or less, more preferably 0.08 mM or more and 1.5 mM or less, further preferably 0.15 mM or more and 1 mM or less, and particularly preferably 0.3 mM or more and 0.8 mM or less. .

In the method for enhancing ethanol production ability of the present invention, any of anaerobic conditions and aerobic conditions can be used as a starter culture method for fermented dairy products, and any of stationary culture method, stirring culture method, and shaking culture method can be used. But it can also be used. At this time, it is not particularly limited as long as necessary microorganisms (such as lactic acid bacteria) contained in the starter for fermented dairy products can grow well, but static culture under aerobic conditions or static culture under anaerobic conditions The method is preferred. And as this culture temperature, L.
From the viewpoint of effectively enhancing the ethanol production ability of fermentum, 20 to 50 ° C is preferable, 30 to 45 ° C is more preferable, 35 to 45 ° C is further preferable, and 37 to 45 ° C is particularly preferable.
Moreover, as this culture | cultivation time, 3-30 hours are preferable from the viewpoint similar to the said viewpoint, 4-20 hours are more preferable, 5-15 hours are further more preferable, and 6-10 hours are especially preferable.

In the starter for fermented dairy products containing L. fermentum with enhanced ethanol production ability of the present invention, it can be used as a main starter in the same manner as usual or conventional ones. At this time, the ethanol production ability of the present invention is enhanced.
Fermentum can be used alone or in combination with microorganisms (such as lactic acid bacteria) contained in normal and conventional main starters for fermented dairy products such as natural cheese and yogurt. You can also. As the main starter to be combined, for example, natural cheese, L.
delbrueckii subsp.bulgaricus species, L.
delbrueckii subsp. lactis species, L.
helveticus species, Lactococcus lactis subsp. cremoris species, Lc. lactis subsp. lactis species, S. thermophilus species, Leuconostoc species, etc., and if yogurt, L. delbrueckii
Subsp. bulgaricus species and S. thermophilus species. In addition, the L.
In the starter for fermented milk products containing fermentum, it can be used not only as a main starter but also as an adjunct starter for the purpose of changing (enhancing, etc.) the flavor and quality of the fermented milk product. At this time, the ethanol production ability of the present invention is enhanced.
Fermentum can be used alone or in combination with microorganisms (such as lactic acid bacteria) contained in normal or conventional adjunct starters for fermented dairy products such as natural cheese and yogurt. You can also. As an adjunct starter to be combined, for example, L.
Examples include casei, L. plantarum, L. rhamnosus, and L. zeae.

  In the method for producing a fermented dairy product that increases the ethanol content of the present invention, L. fermentum of the present invention with enhanced ethanol production ability is used as a starter for the fermented dairy product, and the raw milk is inoculated with the starter. It is characterized by culturing, fermentation and / or aging. Here, a fermented milk product having a high ethanol content can be provided. As a result, a fermented milk product having an enhanced pineapple-like fruity aroma can be provided. In the present invention, the “raw milk” is any raw milk for normal or conventional fermented milk products, mainly made from animal milk such as cattle, sheep and goats, and plant milk such as soy milk. However, raw milk containing skim milk, skim milk, full fat concentrated milk, skim concentrated milk, full fat milk powder, skimmed milk powder, cream, butter and the like can be desirably used. At this time, in the raw milk, the solid content concentration is preferably 8 to 20% by weight, more preferably 9 to 15% by weight, and further preferably 10 to 12% by weight.

  The fermented dairy product manufactured using the starter for fermented dairy products containing L. fermentum with enhanced ethanol production ability of the present invention is intended for normal and conventional fermented dairy products, for example, natural Examples include cheese, yogurt (fermented milk), lactic acid bacteria beverages, and fermented butter.

Moreover, the present invention is characterized by inoculating a raw milk with a starter for fermented dairy products containing L. fermentum having enhanced ethanol-producing ability of the present invention, and culturing and / or fermenting it. It is also a method of increasing the ethanol content of dairy products. And in the method for increasing the ethanol content of the fermented dairy product of the present invention, the ethanol production ability of the present invention is enhanced as a starter for fermented dairy products.
The fermentum is used, and a starter is inoculated into raw milk and cultured and / or fermented. Here, a fermented milk product having a high ethanol content can be provided. As a result, a fermented milk product having an enhanced pineapple-like fruity aroma can be provided.

Furthermore, the present invention is also a fermented dairy product containing L. fermentum whose ethanol production ability is enhanced by the method for increasing the ethanol content of the fermented dairy product of the present invention. And the ethanol production ability was enhanced by the method for increasing the ethanol content of the fermented milk product of the present invention.
In fermented milk products containing fermentum, fermented milk products that are normal or similar to conventional ones are targeted, and examples include natural cheese, yogurt (fermented milk), lactic acid bacteria beverages, and fermented butter.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited by this. In addition, L. fermentum OLL203697 used in the examples, on October 5, 2011 (consignment date), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (1-1-1, Higashi Tsukuba, Ibaraki Prefecture) In the center center 6), FERM with the accession number
BP-11433 is an internationally deposited lactic acid bacterium based on the Budapest Treaty.

[Test Example 1]
Growth of L. fermentum OLL203697 (Accession No. FERM BP-11433) in the starter and L. in model cheese
The effect of yeast extract on ethanol production of fermentum was examined.

(Method)
1. Preparation of L. fermentum starter
SM medium (reduced skim milk medium (solid content concentration: about 10% by weight)) and YE medium (SM medium supplemented with yeast extract to a concentration of 0.5% by weight in the same manner as MRS medium) were prepared. And L. activated culture in MRS medium.
Fermentum OLL203697 (Accession No. FERM BP-11433) was inoculated at 1% by weight, and cultured under anaerobic conditions at 37 ° C. until the late logarithmic growth phase to prepare SM starters and YE starters. In addition, said L.
A culture solution of fermentum OLL203697 was inoculated at 1% by weight in MRS medium and cultured overnight at 37 ° C. under aerobic conditions to prepare an MRS starter (positive control). At this time, L in each starter.
While measuring the number of fermentum bacteria, each starter was used for the production of model cheese.

2. Production of model cheese Reduced skim milk (solid concentration: about 10% by weight) simulating raw milk for cheese is inoculated with a commercially available cheese production starter, and each of the MRS starter, SM starter, YE starter was inoculated. Here, the MRS starter is inoculated at 1% by weight, and the SM starter and YE starter are 1% by weight of the MRS starter and L.
Fermentum OLL203697 was inoculated at a concentration such that the number of bacteria was equivalent. And after inoculating each starter, it fermented until it became pH 5.0 at the temperature (about 37 degreeC) suitable for a commercially available starter. After this fermentation is finished, after removing whey by centrifugation, salt is added to 2% by weight and ripened at 18 ° C. for 2 weeks, model cheese MRS cheese (positive control), SM cheese and YE cheese were produced. Also, L.
A model cheese Control cheese (negative control) without fermentum was also produced. At this time, the ethanol content was measured on the start date and the end date of the ripening, and the ethanol production amount (= ethanol increase amount = ethanol content on the ripening end date−ethanol content on the ripening start date) was calculated. Table 1 shows the model cheese sample names and starter combinations.

(result)
Table 2 shows the amount of ethanol produced (g) in 1 kg of model cheese produced by inoculating a starter prepared by culturing in each medium. From the comparison between SM cheese and YE cheese, it was found that the ethanol production in the model cheese increased by about 3 times. From the above, yeast extract is L. in the starter.
In addition to improving fermentum growth, the model cheese (fermented dairy product) produced using the starter (L.
It was found that fermentum's ability to produce ethanol was enhanced. However, it was also found that adding yeast extract to the milk raw material medium is not sufficient for L. fermentum in model cheese to produce ethanol at 0.5 g / kg or more.

[Test Example 2]
The effect of the alternative components of the MRS medium on the growth of L. fermentum OLL203697 (Accession No. FERM BP-11433) in the starter was examined.

(Method)
Examples of components of the MRS medium include beef extract, Tween 80, magnesium salt, and manganese salt. About these structural components, it substituted by the alternative component (raw material) which can be applied in actually manufacturing fermented milk products, and the alternative component was added (formulation) to the milk raw material culture medium. Specifically, fish meat extract (Fish), Tween 80 (Tw80), magnesium chloride (Mg), and manganese yeast (Mn) were each added to the YE medium. Here, ALL medium (YE medium to which all four components of Fish, Tw80, Mg, and Mn are added), ΔFish medium (YE medium to which three components of Tw80, Mg, and Mn are excluded), ΔTw80 medium (Tw80) YE medium supplemented with 3 components of Fish, Mg, and Mn), ΔMg medium (YE medium supplemented with 3 components of Fish, Tw80, and Mn), ΔMn medium (Fish, Tw80, and Mg excluding Mn) (YE medium supplemented with three components) was prepared (YE medium obtained by removing one component from each of the four components Fish, Tw80, Mg, and Mn was expressed as "Δ" component name "medium). And L. activated culture in MRS medium.
fermentum OLL203697 (Accession No. FERM BP-11433) each inoculated at 1 wt%, cultured under aerobic conditions at 37 ° C until late in the logarithmic growth phase, ALL starter, ΔFish starter, ΔTw80 starter, ΔMg starter and ΔMn starter were prepared. At this time, the number of L. fermentum bacteria in each starter was measured, and the following formula was applied to determine the L.
The contribution rate of each component to the growth of L. fermentum was calculated by comparing the number of bacteria of fermentum and the number of bacteria of L. fermentum in the “Δ” component name ”medium.
Contribution rate (%) = [1− (the number of bacteria with “Δ” component name ”” medium) ÷ (the number of bacteria in ALL medium)] × 100
Table 3 shows the combination of each medium name and alternative components (materials) added to the YE medium. In Table 3, “%” of the component name indicates% by weight, “◯” in the table indicates that the component was used, and “−” indicates that it is not used.

(result)
Table 4 shows the contribution ratios of the alternative components of each MRS medium to the growth of L. fermentum. Here, Fish (fish extract) and Tw80 (Tween) out of the four ingredients Fish, Tw80, Mg and Mn
80), the contribution ratio of Mn (manganese salt) was the largest. In addition, L. cultured in ALL medium.
The number of bacteria in fermentum is larger than the number of bacteria in L. fermentum cultured in SM medium, and L. cultured in MRS medium.
It was found that the four components of Fish, Tw80, Mg, and Mn contributed to the growth of L. fermentum because the number was the same as that of fermentum.

[Test Example 3]
The effect of yeast extract concentration on the growth of L. fermentum OLL203697 (Accession No. FERM BP-11433) in the starter was examined.

(Method)
Various concentrations of yeast extract were added to the Mn medium (SM medium added with manganese yeast to a concentration of 0.2% by weight). Here, the yeast extract was added to the Mn medium so as to have concentrations of 0, 0.05, 0.1, 0.25, and 0.5% by weight, and YE & Mn mediums having respective yeast extract concentrations were prepared. And L. activated culture in MRS medium.
Fermentum OLL203697 (Accession No. FERM BP-11433) was inoculated at 1 wt%, and cultured under aerobic conditions at 37 ° C. until the late logarithmic growth phase to prepare each YE & Mn starter. At this time, the number of L. fermentum bacteria in each starter was measured.

(result)
The number of L. fermentum bacteria in the starter prepared by culturing in each medium is shown in FIG. Here, as the concentration of yeast extract decreases, L.
The number of fermentum bacteria decreased. On the other hand, when the concentration of yeast extract is 0.25 wt%, when 0.5 wt%, L.
It was predicted that the number of bacteria in fermentum was about the same, the concentration of yeast extract was 0.25% by weight or more, and the number of bacteria in L. fermentum remained at the same level. From the curve of the number of bacteria in L. fermentum, when the yeast extract concentration is 0.2% by weight or less, the number of bacteria is 1 × 10 ^9.
It was shown to be below cfu / g.

[Test Example 4]
The effect of manganese concentration on the growth of L. fermentum OLL203697 (Accession No. FERM BP-11433) in the starter was examined.

(Method)
Various concentrations of manganese yeast as manganese were changed and added to the YE medium (SM medium supplemented with yeast extract to a concentration of 0.25% by weight as in the MRS medium). Here, manganese yeast was added to the YE medium so as to have concentrations of 0, 0.05, 0.1, and 0.2% by weight to prepare respective YE & Mn mediums. And L. fermentum activated in MRS medium
Each culture solution of OLL203697 (Accession No. FERM BP-11433) was inoculated at 1% by weight and cultured under aerobic conditions at 37 ° C. until the late phase of the logarithmic growth phase to prepare YE & Mn starters having respective manganese concentrations. At this time, the number of L. fermentum bacteria in each starter was measured.

(result)
The number of L. fermentum bacteria in the starter prepared by culturing in each medium is shown in FIG. Here, as the concentration of manganese yeast decreases, L.
The number of fermentum bacteria decreased. On the other hand, when the concentration of manganese yeast is 0.05 wt%, 0.1 wt% or 0.2 wt%, L.
We were able to predict that the number of fermentum bacteria would remain at the same level. From the curve of the number of bacteria of L. fermentum, when the concentration of manganese yeast is less than 0.05% by weight, the number of bacteria is 1 × 10 ^9.
It was shown to be below cfu / g.

[Test Example 5]
The influence of yeast extract and manganese yeast on the growth of L. fermentum OLL203697 (Accession No. FERM BP-11433) in the starter was examined.

(Method)
Yeast extract and manganese yeast were added in various combinations to the SM medium. Here, in addition to SM medium, YE medium (SM medium added with yeast extract to a concentration of 0.5% by weight as in MRS medium), YE & Mn medium (0.5% by weight yeast extract and 0.2% by weight manganese yeast) SM medium added to a concentration of 2%) and Mn medium (SM medium added with manganese yeast to a concentration of 0.2% by weight). And L. activated culture in MRS medium.
fermentum OLL203697 (Accession No. FERM BP-11433) was inoculated at 1% by weight, and cultured under aerobic conditions at 37 ° C until the late logarithmic growth phase. SM starter, YE starter, YE & Mn starter, An Mn starter was prepared. At this time, L in each starter.
The number of fermentum bacteria was measured. Table 5 shows combinations of the names of the respective media and the components (materials) added to the SM medium. In Table 5, “%” of the component name indicates% by weight, “◯” in the table indicates that the component was used, and “−” indicates that it is not used.

(result)
Table 6 shows the number of L. fermentum bacteria in the starters prepared by culturing in each medium. From the comparison of SM starter and Mn starter, it was found that the growth of L. fermentum was not improved even if manganese yeast alone was added (mixed) to the milk raw material medium at a predetermined concentration. On the other hand, from the comparison of SM starter and YE & Mn starter, when yeast extract and manganese yeast are added (mixed) to milk raw material medium at a predetermined concentration at the same time, L.
It was found that fermentum's growth was improved and a synergistic effect was obtained by the combined use of yeast extract and manganese yeast.

[Test Example 6]
L. fermentum OLL203697 in the starter with manganese yeast addition (Accession number FERM)
The effect of individual components (manganese and yeast) on the growth of BP-11433) was investigated.

(Method)
Regarding the influence of manganese yeast on the growth of L. fermentum, the effects of its individual components manganese and yeast were examined. Here, the above-mentioned YE & Mn medium and YE & inorganic Mn medium (SM medium supplemented with manganese sulfate so that the concentration of yeast extract was 0.5% by weight and manganese yeast was equivalent to 0.2% by weight) were prepared. And L. activated culture in MRS medium.
Fermentum OLL203697 (Accession No. FERM BP-11433) was inoculated at 1% by weight, and cultured at 37 ° C under aerobic conditions until late in the logarithmic growth phase to prepare YE & Mn starters and YE & inorganic Mn starters did. At this time, L in each starter.
The number of fermentum bacteria was measured.

(result)
When the number of L. fermentum bacteria in the starters prepared by culturing in each medium was compared, there was no difference in the number of bacteria between the YE & Mn starter and the YE & inorganic Mn starter. That is, L.
It was confirmed that the influence of manganese yeast on fermentum growth was based on manganese, not yeast (data not shown).

[Test Example 7]
The effect of manganese yeast on the ester productivity of L. fermentum in model cheese was verified.

(Method)
1. Preparation of L. fermentum starter
SM medium (reduced skim milk medium (solid content concentration: about 10% by weight)), Mn medium (SM medium with manganese yeast added to a concentration of 0.2% by weight), YE medium (yeast extract as in MRS medium) SM medium supplemented with a concentration of 0.5% by weight) and YE & Mn medium (SM medium supplemented with 0.5% by weight of yeast extract and 0.2% by weight of manganese yeast). And L. activated culture in MRS medium.
fermentum OLL203697 (Accession No. FERM BP-11433) is inoculated at 1% by weight, and cultured under aerobic conditions at 37 ° C until the late logarithmic growth phase. SM starter, Mn starter, YE starter, And a YE & Mn starter was prepared. At this time, L in each starter.
While measuring the number of fermentum bacteria, each starter was used for the production of model cheese.

2. Production of model cheese Reduced skim milk (solid content concentration: about 10% by weight) simulating raw milk for cheese is inoculated with a commercially available starter for cheese manufacture, SM starter, Mn starter, YE starter, and Inoculated with each of the YE & Mn starters. Here, these inoculation amounts are L.
Fermentum OLL203697 was inoculated at such a ratio that the number of bacteria was equivalent. And after inoculating each starter, it fermented until it became pH 5.0 at the temperature (about 37 degreeC) suitable for a commercially available starter. After this fermentation is finished, after removing whey by centrifugation, salt is added to 2% by weight and aged at 18 ° C for 2 weeks, SM model cheese, Mn cheese, YE Cheese and YE & Mn cheese were produced. At this time, L. in the model cheese on the ripening start date (ripening day 0) and the ending date (after 2 weeks of ripening).
The number of fermentum bacteria was measured, and the difference in the growth rate due to the difference in the composition of the starter was compared. Furthermore, the content of ethyl butyrate after 2 weeks of aging was measured as an indicator of a pineapple-like fruity scent. The method for measuring the amount of ethyl butyrate is as follows.
0.5 g of sample was collected in a vial, 4.5 g of saturated saline was added and sealed, and then kept at 60 ° C. for 40 minutes, and ethyl butyrate volatilized in the headspace was replaced with solid-phase microfiber (SUPELCO,
Stable Flex (DVB / Carboxen / PDMS; 50 / 30μ \ ochm)) was extracted. The fiber was introduced into GC / MS and subjected to Scan mode measurement. From the obtained chromatogram, the peak area of m / z88 ions was taken as the detection amount.

(result)
L. fermentum on the 0th day of ripening (at the start of ripening) and 2 weeks after ripening in model cheese produced by inoculating each starter
Table 7 shows the number of OLL203697 bacteria and their multiplication factor. The amount of ethanol and ethyl butyrate (area value) in the model cheese after 2 weeks of ripening, the amount of ethanol produced per fixed number of L. fermentum OLL203697, and butyric acid The amount ratio of ethyl is shown in Table 8. L during the ripening period of model cheese.
The growth rate of fermentum OLL203697 was about 2 times for any model cheese, and 2.1 times for YE & Mn cheese, which was lower than other model cheeses. However, the amount of ethanol produced and the amount of ethyl butyrate (area value) in YE & Mn cheese were significantly higher than in other model cheeses, and the production ratio per fixed number of bacteria was also significantly higher. From this, when yeast extract and manganese yeast are added (mixed) to the milk raw material medium at a predetermined concentration at the same time, L.
The ethanol production ability per cell of fermentum was significantly enhanced, and the remarkable effect of significantly increasing the ester (ethyl butyrate) involved in cheese aroma was revealed.

  According to the present invention, since the amount of ethanol produced by L. fermentum in fermented dairy products is increased, fermented dairy products having a high ethanol content including natural cheese can be provided. And as a result, fermented milk products which strengthened fruity fragrance like pineapple like natural cheese can be provided.

Claims (7)

L. fermentum is cultured using a milk raw material medium to which a nitrogen source and / or a divalent metal ion is added as a pre-culture medium for a starter for fermented dairy products. L.
A method for enhancing the ethanol production ability of fermentum. The method for enhancing ethanol-producing ability according to claim 1, wherein the milk raw material medium comprises skim milk, skim concentrated milk, and reduced skim milk having a solid content concentration of 8 to 12%. The method for enhancing ethanol-producing ability according to claim 1, wherein the nitrogen source is a yeast extract and / or a peptide derived from milk. The divalent metal ion is at least one or more of Fe ²⁺ , Cu ²⁺ , Zn ²⁺ , Ca ²⁺ , Mg ²⁺ and Mn ²⁺ , according to claim 1, The method for enhancing ethanol production ability as described. A starter for fermented dairy products containing L. fermentum whose ethanol production ability is enhanced by the method for enhancing ethanol production ability according to claims 1 to 4. A method for producing a fermented dairy product with an increased ethanol content, wherein the raw milk is inoculated with the starter according to claim 5 and cultured, fermented and / or aged. A fermented dairy product having a high ethanol content produced by the method for producing a fermented dairy product according to claim 6.

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