JP7000012B2 - Yeast screening method and method for producing fermented cacao raw materials - Google Patents

Description

The present invention relates to a method for screening yeast and a method for producing a fermented product of cacao raw material using yeast selected by the screening method.

It is known that the flavor of chocolate is influenced by the flavor of cocoa mass, which is its main ingredient. Cocoa mass is prepared based on the natural fermented pulp (cocoa pulp) and seeds (cocoa beans) taken from the cocoa pod. However, in natural fermentation, the flavor of the fermented product may change due to changes in the environment and fermentation conditions, and this change in flavor may affect the quality of the final products such as chocolate and cocoa.

Patent Document 1 discloses a technique in which a cacao raw material such as cacao mass is enzymatically treated to produce an enzyme-treated product having a strong umami taste, and the processed product is blended into foods and drinks such as chocolate. Further, in Patent Document 2, an enzyme-treated product of cacao raw material such as cacao mass is fermented with lactic acid bacteria and yeast to produce a cacao-processed product having a unique fermented flavor, and the processed product is blended in foods and drinks such as chocolate. The technology is disclosed.

Japanese Unexamined Patent Publication No. 2007-43931 Japanese Unexamined Patent Publication No. 2010-51250

However, all of the above techniques are premised on the use of cacao raw materials obtained by natural fermentation, and the problems of flavor change and adverse effects on quality due to natural fermentation have not yet been solved.

It is an object of the present invention to provide a screening method for selecting yeast that produces a fermented product having a good flavor. It is also an object of the present invention to provide a method for producing a fermented product of a cocoa raw material having a good flavor.

According to the present invention, the following inventions are provided.
[1] A screening method for screening yeast that produces a fermented product having a desired flavor characteristic, which comprises culturing a test yeast in the presence of a cocoa mass extract.
[2] The screening method according to the above [1], wherein the desired flavor characteristics are different from the flavor characteristics of the separation source of the test yeast.
[3] The screening method according to the above [1] or [2], wherein the desired flavor characteristic is a fruit scent, a brewed liquor scent, a distilled liquor scent or a floral scent.
[4] The test yeast was selected from the group consisting of yeasts of the genus Devaliomyces, yeasts of the genus Kluyberomyces, yeasts of the genus Saccharomyces, yeasts of the genus Zygosaccharomyces, yeasts of the genus Pikia, yeasts of the genus Candida and yeasts of the genus Hansenia spora. The screening method according to any one of [1] to [3] above, which is yeast.
[5] Yeast selected by the screening method according to any one of [1] to [4] above.
[6] A method for producing a fermented product, which comprises adding the yeast according to the above [5] to a cacao raw material and fermenting it.
[7] The production method according to the above [6], wherein the yeast belongs to Kluyveromyces lactis, Devariomyces Hanseni, Kluyveromyces marcianus or Saccharomyces cerevisiae.
[8] The production method according to the above [6], wherein the cacao raw material is selected from the group consisting of cacao beans, cacao pulp, and processed products thereof.
[9] The production method according to the above [8], wherein the processed product is cacao pulp juice, cacao mass and cacao mass extract.
[10] The production method according to the above [6], wherein the cacao raw material contains cacao beans and the fermented product is fermented cacao beans.
[11] A cocoa mass, which comprises preparing cocoa mass using the fermented cocoa beans prepared by the production method according to the above [10] as a raw material, and in some cases preparing cocoa or cocoa butter from the cocoa mass. How to make cocoa or cocoa butter.
[12] An oil-based confectionery or a cocoa-blended product using the fermented cocoa beans prepared by the production method according to the above [10] or the cocoa mass, cocoa or cocoa butter prepared by the production method according to the above [11] as a raw material. A method for producing an oil-based confectionery or cocoa-blended product, which comprises preparing.
[13] The production method according to the above [6], wherein the cacao raw material is cocoa mass and / or cocoa mass extract, and the fermented product is fermented cocoa mass and / or fermented cocoa mass extract.
[14] The production method according to the above [13], further comprising preparing an extract of fermented cocoa mass from fermented cocoa mass.
[15] Cocoa and / or the fermented cocoa mass and / or the fermented cocoa mass extract prepared by the production method according to the above [13], and / or the fermented cocoa mass extract prepared by the production method according to the above [14]. / Or a method for producing cocoa and / or cocoa butter, comprising preparing cocoa butter.
[16] Fermented cocoa mass and / or fermented cocoa mass extract prepared by the production method according to the above [13], and / or fermented cocoa mass extract prepared by the production method according to the above [14], and /. Alternatively, a method for producing an oil-based confectionery or a cocoa-blended product, which comprises preparing an oil-based confectionery or a cocoa-blended product using cocoa and / or cocoa butter prepared by the production method according to the above [15] as a raw material.
[17] The production method according to the above [6], wherein the cacao raw material is cacao pulp and / or cacao pulp juice, and the fermented product is fermented cacao pulp and / or fermented cacao pulp juice.
[18] The production method according to the above [17], further comprising squeezing the fermented cocoa pulp to prepare a juice of the fermented cocoa pulp.
[19] To the fermented cocoa pulp and / or the fermented cocoa pulp juice prepared by the production method according to the above [17], and / or the juice of the fermented cocoa pulp prepared by the production method according to the above [18]. A method for producing flavored cocoa beans, cocoa nibs and / or cocoa mass, comprising immersing the cocoa beans, cocoa nibs and / or cocoa mass.
[20] Cocoa mass is prepared using the flavored cocoa beans and / or cocoa nibs prepared by the production method according to the above [19] as a raw material, and in some cases, cocoa and / or cocoa butter is prepared from the cocoa mass. A method for producing cocoa mass, cocoa and / or cocoa butter, comprising:
[21] A method for producing cocoa and / or cocoa butter, which comprises preparing cocoa and / or cocoa butter from flavored cocoa butter prepared by the production method according to the above [19].
[22] Flavored cocoa beans, cocoa nibs and / or cocoa mass prepared by the production method according to the above [19], and / or cocoa mass, cocoa and / or cocoa mass prepared by the production method according to the above [20]. / Or cocoa butter, and / or cocoa and / or cocoa butter prepared by the production method according to the above [21], which comprises preparing an oily confectionery or a cocoa-blended product using cocoa butter as a raw material. Manufacturing method for cocoa-blended products.

According to the screening method of the present invention, yeast that produces a fermented product having a good flavor can be easily and inexpensively selected. Further, according to the production method of the present invention, a fermented product of a cacao raw material having a good flavor can be prepared while controlling the bacterial flora. Therefore, according to the present invention, it is advantageous that cocoa mass and the like having a good flavor can be prepared, and thus chocolate, cocoa and cocoa butter having a good flavor can be stably prepared.

Specific description of the invention

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for screening yeast that produces a fermented product having a desired flavor characteristic. In the screening method of the present invention, the test yeast is cultured in the presence of a cocoa mass extract. The cocoa mass extract can be prepared, for example, as follows. That is, the cocoa mass obtained according to a conventional method can be extracted with a solvent (preferably water or hot water), and the supernatant obtained by solid-liquid separation of the extract can be used as the cocoa mass extract. As such a cocoa mass extract, a commercially available one can be used.

In order to efficiently select the test yeast, the concentration of the cacao extract extract in the culture medium can be, for example, 1 to 30% by mass (in terms of solid content), preferably 3 to 10% by mass. (Solid content conversion). In order to efficiently select the test yeast, saccharides such as monosaccharides and disaccharides can be added to the culture medium, and the concentration of the saccharides added in that case is 0.1 to 18% by mass. It can be, preferably 0.5 to 10% by mass. Examples of the saccharides that can be added include monosaccharides such as glucose, fructose, galactose, xylose, and arabinose, and disaccharides such as sucrose, maltose, lactose, and cellobiose. The above may be used in combination.

In addition to cocoa extract and saccharides, components used for yeast culture can be added to the culture medium used in the screening method of the present invention, and such components include yeast extract, malt extract, and peptone. Can be mentioned. If necessary, trace metals such as iron, zinc, copper and manganese, and vitamins such as biotin may be further added to the culture medium.

Yeast fermentation in the culture medium can be carried out by inoculating the yeast grown by the preculture into the main culture medium and performing the main culture. The conditions of the main culture are not particularly limited, but the culture can be carried out under the conditions of, for example, a culture temperature of 7 to 40 ° C. and a culture time of 6 to 168 hours. The number of yeast cells per main culture medium is not particularly limited, but for example, seeding can be carried out so as to be 1 × 10 ⁴ to 3 × 10 ⁸ per 1 ml of the medium.

As shown in Examples below, the screening method of the present invention can detect the flavor of the fermented product of the test yeast with high sensitivity, and therefore, the fermented product having the flavor characteristics different from the flavor characteristics of the separation source of the test yeast can be detected. The yeast to be produced can be selected. Further, according to the screening method of the present invention, yeast that produces a fermented product of fruit aroma can be selected, and further, the aroma of brewed sake such as wine and sake, and aged distillation such as rum and whiskey can be selected. Yeasts with a scent of sake and floral scents such as jasmine and tin orchid can be selected. In particular, according to the screening method of the present invention, fermented products of fruit scents can be detected, and yeasts that produce these scents during cacao fermentation can be accurately selected.

The yeast used for the screening method is not particularly limited, and for example, yeast of the genus Debaryomyces , yeast of the genus Kluyveromyces , yeast of the genus Saccharomyces , and yeast of the genus Zygosaccharomyces . , Yeasts selected from the group consisting of yeasts of the genus Pichia , yeasts of the genus Candida and yeasts of the genus Hanseniaspora can be used in the screening method of the present invention.

The yeast selected by the screening method of the present invention can be used for fermentation of cacao raw materials. That is, according to the present invention, there is provided a method for producing a fermented product of a cacao raw material, which comprises adding yeast selected by the screening method of the present invention to the cacao raw material and fermenting the yeast.

Yeasts that can be used in the production method of the present invention include yeasts that produce fermented fruit scents, yeasts that produce fermented scents of brewed liquor, yeasts that produce fermented scents of distilled liquor, or fermentation of floral scents. Examples include yeast that produces products. Yeasts that produce fermented fruit scents include yeasts of the genus Debaryomyces (eg, yeasts of the genus Debaryomyces hansenii ) and yeasts of the genus Kluyveromyces (eg, Kluyveromyces ). Yeast belonging to Kluyveromyces lactis , yeast belonging to Kluyveromyces marxianus ) can be used.

The yeast that can be used in the production method of the present invention also belongs to Kluyveromyces marxianus , a yeast belonging to Debaryomyces hansenii , a yeast belonging to Kluyveromyces lactis . Yeasts include yeasts belonging to Saccharomyces cerevisiae , and one of a plurality of yeast strains belonging to these may be used alone or in combination of two or more. Preferred yeast strains include Kluyveromyces marxianus NBRC0272 strain , Saccharomyces cerevisiae NBRC0850 strain , Kluyveromyces marxianus NBRC1777 strain , Kluyveromyces lactis var . Included are marxianus JCM21874, Kluyveromyces marxianus JCM21878 and Kluyveromyces lactis JCM22014, more preferably Kluyveromyces lactis JCM5219, Debaryomyces hansenii JCM5912 and Kluyveromyces lactis var. In the production method of the present invention, preferably one or more isolated yeast strains can be added and used.

Here, the strains whose reference numbers start with "JCM" are stored in the following storage institutions and can be provided by application.
Preservation organization name: RIKEN BioResource Center Microbial Material Development Office (Contact: Postal code 351-0198, 2-1 Hirosawa, Wako City, Saitama Prefecture, Phone number 048-467-9560)

In addition, in this specification, strains whose reference number starts with "NBRC" are stored in the following depository institutions and can be provided by application.
Depositary organization name: Biotechnology Center, Biotechnology Center, Incorporated Administrative Agency Biotechnology Center (Contact: Postal code 292-0818, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Phone number 0438-20-5763 )

Examples of the cacao raw material as a fermentation raw material in the production method of the present invention include cacao beans and cacao pulp which are the contents of the cacao pod, and processed products thereof are also included. Examples of the processed cocoa beans and cocoa pulp include cocoa pulp juice obtained by pressing cocoa pulp, cocoa mass obtained by processing cocoa beans according to a conventional method, and an extract of the cocoa mass. Examples of the cocoa mass extract include an extract obtained from cocoa mass by an extraction treatment using a solvent such as water or hot water.

Hereinafter, the production method of the present invention will be described for each cacao raw material which is a fermentation raw material.
<When fermenting the contents of the cacao pod>
In the production method of the present invention, cocoa beans or cocoa pod contents can be fermented. In this case, the fermented product prepared is fermented cocoa beans or fermented cocoa pod contents. When the contents of the cocoa pod are fermented, the cocoa pulp is also subjected to yeast fermentation in addition to the cocoa beans, and the flavor produced by the fermentation of the cocoa pulp is given to the cocoa beans. It is preferable to let them.

In the production method of the present invention, when the cacao beans or the contents of the cacao pod are fermented with yeast, a raw material other than the cacao raw material may be added and fermented together. By adding fruit pulp or juice as a raw material other than the cacao raw material, the flavor produced by the fermentation of the fruit can be imparted to the cacao beans. Further, when molasses is used as a raw material other than the cacao raw material, the flavor produced by the fermentation of the molasses can be imparted to the cacao beans. Ingredients other than the cacao ingredient may be yeast-fermented in advance before being added to the cacao pod contents.

Yeast fermentation can be carried out by inoculating the cacao raw material with yeast grown by preculture. When the cacao raw material is the cacao pod contents, yeast can be inoculated into the cacao pod contents obtained by collecting the contents immediately after harvesting the cacao pods. The cocoa pod contents may be those recovered after pod storage for several days after harvest, or from cocoa pods stored after harvest under low temperature conditions such as refrigeration or freezing. good. Fermentation may be carried out under aerobic conditions or under anaerobic conditions by nitrogen substitution. During fermentation, it may be left standing or may be stirred as appropriate.

The conditions for yeast fermentation are not particularly limited, but the yeast fermentation can be carried out under the conditions of, for example, a culture temperature of 7 to 40 ° C. and a culture time of 6 to 168 hours. The number of yeast cells per cacao raw material is not particularly limited, but for example, seeding can be carried out so as to be 1 × 10 ⁴ to 3 × 10 ⁸ per 1 g of the raw material.

In the production method of the present invention, the contents of the cacao pod can be aseptically taken out from the cacao pod, and one or more yeast strains selected by the screening method of the present invention can be added for yeast fermentation. Natural fermentation by the conventional method was carried out in an environment close to the open air, and it was difficult to carry out by strictly controlling factors such as the fermentation environment and indigenous bacteria.However, when yeast fermentation is carried out by the production method of the present invention, Cocoa beans having desired aroma characteristics can be prepared while appropriately controlling the progress of yeast fermentation. In particular, it is advantageous to add a blend of a plurality of yeast strains because cocoa beans having a rich flavor and aroma characteristics can be prepared while controlling the bacterial flora.

The production method of the present invention can also be carried out in natural fermentation by a conventional method. That is, yeast fermentation can be carried out by adding one or more yeast strains selected by the screening method of the present invention before the start of natural fermentation of the cacao pod contents or during natural fermentation. Embodiments are also included in the production method of the present invention. In natural fermentation, the flavoring of cacao beans is easily affected by the fermentation environment and the like, but such an embodiment is advantageous in that the desired aroma characteristics can be reliably imparted to the cacao beans.

The obtained cocoa beans can be processed into cocoa mass according to a conventional method. That is, cacao nibs can be obtained by removing the skin from the cacao beans with a separator, roasting the cacao nibs with a roaster, and grinding the roasted cacao nibs with a grinder to prepare cacao mass. The cocoa beans may be roasted and then the skin removed to obtain cocoa nibs. Further, in some cases, a plurality of kinds of cocoa nibs may be mixed by a blender before grinding by a grinder. The obtained cocoa mass can be used as a raw material for oil-based confectionery, cocoa and cocoa butter.

<When fermenting cocoa mass, etc.>
In the production method of the present invention, cocoa mass and / or cocoa mass extract can be fermented. In this case, the fermented product prepared is fermented cocoa mass and / or fermented cocoa mass extract. The prepared fermented cocoa mass may be further subjected to an extraction process to obtain an extract of fermented cocoa mass. The extraction treatment can be carried out using a solvent such as water or hot water. Fermentation of cocoa mass and cocoa mass extract by yeast can be carried out under the same conditions as described above, but in culturing, either shaking culture or static culture may be used, and primary stirring with any of these may be used. It may be carried out in combination. Even if the cocoa mass and its extract to be fermented were prepared without being subjected to the fermentation treatment by the production method of the present invention, they were prepared by being subjected to the fermentation treatment by the production method of the present invention. It may be a thing.

The obtained fermented cocoa mass, the extract of the fermented cocoa mass, and the fermented cocoa mass extract can be used as raw materials for oily confectionery, cocoa, and cocoa butter.

<When fermenting cacao pulp, etc.>
In the production method of the present invention, cocoa pulp and / or cocoa pulp juice can be fermented. In this case, the fermented products prepared are fermented cocoa pulp and fermented cocoa pulp juice. The prepared fermented cocoa pulp may be further subjected to a pressing treatment to obtain a juice of the fermented cocoa pulp. Fermentation of cacao pulp and cacao pulp juice with yeast can be carried out under the same conditions as described above, but in the case of culturing, either shaking culture or static culture may be used, and further, primary stirring with any of these. May be carried out in combination.

In the production method of the present invention, when the cacao pulp and / or the cacao pulp juice is fermented with yeast, a raw material other than the cacao raw material may be added and fermented together. When the pulp or juice of a fruit is added as a raw material other than the cacao raw material, the flavor produced by the fermentation of the fruit can be imparted to the cacao pulp and the cacao pulp juice, and the flavor is imparted to the cacao beans by the dipping step described later. can do. Further, when molasses is added as a raw material other than the cacao raw material, the flavor produced by the fermentation of the molasses can be imparted to the cacao pulp and the cacao pulp juice, and the flavor is imparted to the cacao beans by the dipping step described later. Can be done. Raw materials other than the cacao raw material may be yeast-fermented in advance before being added to the cacao pulp and the cacao pulp juice.

The obtained fermented cocoa pulp and the juice of the fermented cocoa pulp and the fermented cocoa pulp juice can be used for flavoring cocoa beans, cocoa nibs and cocoa mass. The cocoa beans, cocoa nibs and cocoa mass used for flavoring were prepared by being subjected to the fermentation treatment by the production method of the present invention even if they were prepared without being subjected to the fermentation treatment by the production method of the present invention. It may be a thing. Flavoring can be carried out by immersing cocoa beans, cocoa nibs and / or cocoa mass in any or a combination of fermented cocoa pulp and juice of the fermented cocoa pulp and fermented cocoa pulp juice. The immersion can be performed, for example, under the conditions of 2 to 40 ° C. and 4 to 120 hours, but is not limited to this.

Flavored cocoa beans and cocoa nibs can be processed into cocoa mass according to a conventional method, and the cocoa mass can be further processed into cocoa and cocoa butter according to a conventional method. In addition, the flavored cocoa mass can be processed into cocoa and cocoa butter according to a conventional method. Cocoa can be obtained as cocoa cake by separating cocoa butter from cocoa mass with a cocoa press. The obtained cocoa cake can be crushed by a cocoa mill and processed into cocoa powder. In the present invention, cocoa includes both cocoa cake and cocoa powder.

In the present invention, oil-based confectionery can be prepared using the cocoa mass, cocoa and cocoa butter produced as described above as raw materials. The oil-based confectionery referred to in the present invention is not limited to chocolate, quasi-chocolate, and chocolate products in the fair competition rules regarding the labeling of chocolates, which is a rule approved by the Japan Fair Trade Commission, and does not fall under the above rule. It is used to include all kinds of oil-based confectionery such as temper type and non-temper type fat cream. In the present invention, chocolate is used to include any chocolate such as sweet chocolate, milk chocolate, and white chocolate.

In the production of oil-based confectionery, in addition to cocoa bean-derived raw materials (cocoa mass, cocoa powder, cocoa butter, etc.), sugars, sweeteners, dairy raw materials, oil and fat raw materials, emulsifiers, flavors, coloring agents and the like can be added. Examples of sugars include monosaccharides and disaccharides such as sucrose (sugar, powdered sugar), glucose, fructose, maltose, invert sugar and lactose. Examples of the sweetener include high-sweetness sweeteners such as sucralose and saccharin. Examples of the dairy raw material include whole milk powder, skim milk powder, cheese powder and the like. Examples of the emulsifier include lecithin, glycerin fatty acid ester, and sucrose fatty acid ester. Furthermore, powdered fruits and vegetables, beans (almonds, hazelnuts, sesame, etc.), grains (rice, wheat, etc.), dried fruits (raisins, oranges, etc.), baked confectionery (cookies, biscuits, etc.), etc. You can also combine raisins.

The oil-based confectionery can be prepared according to a conventional method. For example, after mixing the above raw materials, a refining (miniaturization) step, a contouring step, a tempering step, a molding step, and heat resistance are imparted as necessary. It can be manufactured through a storage process for this purpose.

In the present invention, instant cocoa, cocoa beverage, coffee, coffee beverage, soft beverage containing coffee, dairy beverage, refreshing drinking water, etc., using the cocoa mass, cocoa and cocoa butter produced as described above as raw materials according to a conventional method. A product containing cocoa (sometimes referred to as a "cocoa-blended product" in the present invention) can be prepared. The cocoa-blended product of the present invention may contain sugars, sweeteners, dairy raw materials, oil and fat raw materials, salts, emulsifiers, stabilizers, pH adjusters, flavors, coloring agents and the like as auxiliary raw materials.

The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples.

Example 1: Examination of yeast fermentation conditions The yeast Kluyveromyces marxianus JCM21874 strain derived from dairy raw materials is cultivated in a cocoa extract medium, and changes in the pH and Brix value (Bx) of the fermented product as the fermentation progresses are measured and sensory. Evaluation was performed.

(1) Culture conditions 500 μL of JCM21874 strain that had been cryopreserved was inoculated into 50 mL of YPD medium (medium composition: yeast extract 10 g, peptone 20 g, glucose 20 g, distilled water 1 L) and cultured at 30 ° C. for 18 hours with shaking. Yeast was activated. The obtained culture broth was used as a preculture broth and used for the following main culture.

The main culture medium was prepared as follows. The cacao extract (Meiji Co., Ltd.) (hereinafter, simply referred to as "cacao extract") obtained by solid-liquid separation of the hot water extract of cacao mass and concentrating the supernatant is used in three concentrations, that is, cacao extract. The concentrations were adjusted to 100%, 50%, and 25%. The obtained three groups were further divided into two groups depending on the presence or absence of glucose addition, that is, two groups, one without addition and one with 1% by mass of glucose added, and a total of 6 groups of main culture media were prepared. Each group was further divided into four, and a total of 24 main culture media were prepared (sample numbers 1 to 24).

500 μL of the preculture solution was inoculated into 50 mL of each main culture medium and statically cultured at 30 ° C. for 3 days. The main culture solution (fermented cacao extract) was collected every day including the fermentation start date, and pH and Bx were measured and sensory evaluation was performed.

(2) Measurement and measurement results of pH and Bx The pH of the fermented product was measured using a tabletop pH meter F-71 (manufactured by HORIBA, Ltd.). Bx of the fermented product was measured using a pocket sugar content meter PAL-J (manufactured by Atago Co., Ltd.).

Table 1 shows the measurement results of pH and Bx of the fermented cacao extract.

From the results in Table 1, changes in pH and Bx were confirmed as the fermentation progressed in the group to which glucose was added (sample numbers 13 to 24). On the other hand, in the group to which glucose was not added (sample numbers 1 to 12), no significant change in pH and Bx was confirmed.

(3) Sensory evaluation The fermented cacao extract was diluted so that the concentration of the cacao extract was 25%. That is, the fermented product cultured in the medium having a cocoa extract concentration of 100% was diluted 1/4, and the fermented product cultured in the medium having a cocoa extract concentration of 50% was diluted 1/2. Fermented products cultured in a medium having a cocoa extract concentration of 25% were not diluted. 7% by mass of sucrose was added to each fermented product and dissolved to prepare a sample for sensory evaluation. Sensory evaluation was performed by sniffing the scent and tasting by 10 specialized panelists.

It was confirmed that the flavor change due to yeast fermentation was particularly large in the fermented product having 1% by mass of glucose added and the cacao extract concentration of 25%. That is, it was confirmed that the cacao extract medium is a medium capable of sensitively detecting the flavor of the fermented product produced by yeast fermentation.

Example 2: Fermentation test and sensory evaluation of yeast strain The yeast strain was cultured in a cacao extract medium, and the sensory evaluation of the fermented product was performed.

(1) Culture conditions Each 300 μL yeast strain that had been cryopreserved was inoculated into 15 mL of YM medium (medium composition: glucose 10 g, peptone 5 g, yeast extract 3 g, malt extract 3 g, distilled water 1 L, the same applies hereinafter), and 28 Yeast was activated by shaking culture at ° C for 18 hours. The obtained culture broth was used as a preculture broth and used for the following main culture. The yeast strains used in the fermentation test were as shown in Table 2.

As the main culture medium, a cacao extract medium having a cacao extract concentration of 25% and glucose added in an amount of 1% by mass was prepared in the same manner as in Example 1.

Each 3 mL preculture solution was inoculated into 150 mL of each main culture medium and statically cultured at 28 ° C. for 3 days. As a control, a test group was set up in which yeast (preculture solution) was not inoculated into the main culture medium. The main culture solution (fermented cacao extract) was collected and subjected to sensory evaluation.

(2) Sensory evaluation 8% by mass of sucrose was added to the fermented cacao extract and dissolved to prepare a sample for sensory evaluation. The sensory evaluation was carried out by 10 specialized panelists by sniffing the scent and tasting, and using a paper listing various flavor keywords, circle the flavors that felt that there was a difference in flavor from the control, and freely comment. Described. The obtained sensory evaluation results were aggregated for each yeast strain (1 point per panelist). Next, as shown in Table 3, the sensory evaluation results were aggregated for each yeast genus and divided by the number of strains to calculate the average value.

(3) Evaluation results Table 3 shows the results of the sensory evaluation of the fermented cacao extract.

From the values of various flavors in Table 3, it was confirmed that the difference in flavor of the fermented product could be detected by culturing the test yeast in the cacao extract medium. In addition, the following flavors were observed for the fermented product of the test yeast from the comments of the panelists.
Genus Debaryomyces : A fragrant fermented feeling like miso and soy sauce is given. It has a fruity feel like melon and pear.
Genus Kluyveromyces : It has a liquor feeling like wine and sake. It has a fruity flavor.
Genus Saccharomyces : Fermented feeling like lees and pickles.
Zygosaccharomyces genus: does not contribute to flavor.

From the above results, it was shown that yeast that produces a fermented product having the desired flavor characteristics can be selected by culturing the yeast strain in a cacao extract medium and performing sensory evaluation.

Example 3: Fermentation and sensory evaluation of cacao pod contents Fermentation of cacao beans and cacao pulp were fermented using 4 of the yeast strains used in the fermentation test of Example 2, and fermentation evaluation and sensory evaluation of the fermented product were performed. ..

(1) Yeast strains The following four strains were used as yeast.
Kluyveromyces lactis var. Lactis NBRC 1903 strain
Kluyveromyces marxianus JCM21878 strain
Saccharomyces cerevisiae NBRC0244 strain
Saccharomyces cerevisiae JCM1817 strain

(2) Pre-culture Each 300 μL yeast strain that had been cryopreserved was inoculated into 15 mL of YM medium and cultured with shaking at 28 ° C. for 16 hours to activate yeast. The obtained culture broth was used as a preculture broth and used for the following main culture.

(3) Main culture The surface of unfermented Ecuadorian cacao pods was sterilized by spraying 70% ethanol and wiping it off. Cut the cocoa pod with the blade of a sterilized cutter, and remove the cocoa beans and cocoa pulp from the cocoa pod so that each sample has 8 cocoa beans and the total weight of the cocoa beans and cocoa pulp is 20 to 25 g. It was taken out aseptically (in the present specification, the cocoa beans and cocoa pulp taken out from the cocoa pod are sometimes simply referred to as "cocoa pod contents"). This was prepared for 18 samples.

In order to confirm that the above operation is performed aseptically, the removed cacao pod contents are suspended in sterile diluted water, 100 μL of the suspension is applied to a YPD plate medium, and the suspension is applied at 30 ° C. 3 After culturing for a day and performing the initial bacterial count test, no bacterial bacteria were detected, confirming that the operation was aseptic.

Each preculture solution of 0.2 mL or 2 mL was inoculated into the cocoa pod content sample and statically cultured at 30 ° C. for 1 or 3 days. As a control, a test plot was set up in which yeast (preculture) was not inoculated into the cacao pod content sample. This culture (fermented product of cacao pod contents) was collected and subjected to fermentation evaluation and sensory evaluation.

(4) Fermentation evaluation and sensory evaluation The fermented cacao pod contents were collected on the 1st and 3rd days from the start of fermentation and used as samples for evaluation. The sensory evaluation was carried out by sniffing the scent by 10 specialized panelists. Fermentation evaluation was performed visually.

Table 4 shows the results of sensory evaluation of the fermented cacao pod contents.

Fermentation evaluation confirmed that no changes were observed in the sample not inoculated with the yeast strain, while white yeast colonies were observed in the sample inoculated with the yeast strain. Remarkable colonies were confirmed at 2.0 mL from the yeast addition amount of 0.2 mL and on the 3rd day from the 1st fermentation day. From the results of the sensory evaluation, it was confirmed that the cacao extract fermented product obtained in Example 2 and the cacao pod content fermented product obtained in Example 3 had the same sensory evaluation results with respect to the fruit aroma. That is, it was shown that the result of the sensory evaluation in the fermentation test corresponds to the sensory evaluation of the fermented product obtained by the actual fermentation of the cacao raw material with respect to the fruit aroma. Of the yeast strains used for fermentation, the Kluyveromyces lactis var. Lactis NBRC1903 strain had a relatively strong fruit aroma, and was used in the production of chocolate in Example 4.

Example 4: Production of chocolate using selected yeast strains Cocoa beans and cocoa pulp are fermented and fermented using one yeast strain that has a relatively strong fruit aroma among the yeast strains used in the fermentation test of Example 2. Chocolate was produced from the product and subjected to sensory evaluation.

(1) Yeast strain As the yeast strain, Kluyveromyces lactis var. Lactis NBRC 1903 strain was used.

(2) Pre-culture Pre-culture was performed in the same manner as in Example 3.

(3) Main culture The main culture was carried out in the same manner as in Example 3 except that 7 mL of the preculture solution was inoculated into a sample of about 700 g of cacao pod contents and then statically cultured at 30 ° C. for 2 days. As a control, a test plot was set up in which the contents of the cacao pod were not inoculated with yeast (preculture). This culture (fermented cacao pod contents) was collected and used for chocolate production.

(4) Production of chocolate Chocolate was produced according to a conventional method. Specifically, the fermented cacao pod contents were heated and dried at 50 ° C. for 3 days using a dry oven. After roasting the dried cocoa pod contents fermented product, the cocoa pulp and cocoa bean seed coats were removed to obtain cocoa nibs. Cacao nibs were ground with a roll mill to obtain cocoa mass. 50.0 parts by mass of cocoa mass, 37.5 parts by mass of sugar, 12.0 parts by mass of cocoa butter, and 0.5 parts by mass of lecithin were mixed, melted in a water bath, molded, and cooled and solidified to obtain sweet chocolate. ..

(5) Sensory evaluation The produced chocolate was used as a sample for evaluation. Sensory evaluation was performed by sniffing the scent and tasting by 10 specialized panelists. A fruit flavor was observed in chocolate produced from a fermented cacao pod content fermented using a yeast strain, which was consistent with the sensory evaluation results of the fermented cacao extract obtained in Example 2.

Example 5: Selection of yeast strains A plurality of yeast strains were cultured in a cacao extract medium, and the fermented product was subjected to sensory evaluation to select yeasts that produced a fermented product having a good flavor.

(1) Yeast strains The strains shown in Table 5 were used as yeast.

(2) Culturing conditions Culturing was carried out under the same conditions as in Example 2 except that the fermentation days were set to 2 days.

(3) Sensory evaluation 8% by mass of sucrose was added to the fermented cacao extract and dissolved to prepare a sample for sensory evaluation. Sensory evaluation was performed by sniffing the scent and tasting by 10 specialized panelists. The results are as shown in Table 5.

From the results shown in Table 5, it was confirmed that the flavor of the fermented cacao extract differs greatly depending on the yeast strain, and that the yeast strain that produces the fermented product having a flavor characteristic different from that of the separation source can be discriminated. In addition, yeast strains that produce fermented products with fruity aromas such as grapes, bananas, pears, pine, melons, watermelons, and apples are grouped with yeast strains that exhibit generally unfavorable aromas such as pickles and medicines. It was confirmed that it could be identified separately. Specifically, as yeast strains that produce fermented products with fruit aroma, Kluyveromyces marxianus NBRC0272 strain, Saccharomyces cerevisiae NBRC0850 strain, Kluyveromyces marxianus NBRC1777 strain, Kluyveromyces lactis var . , Kluyveromyces lactis JCM5219 strain, Debaryomyces hansenii JCM5912 strain, Kluyveromyces marxianus JCM21874 strain, Kluyveromyces marxianus JCM21878 strain, Kluyveromyces lactis JCM2 2014 strain could be identified. In particular, it was confirmed that the fermented cacao extract from the Kluyveromyces lactis JCM5219 strain and the fermented cacao extract from the Debaryomyces hansenii JCM5912 strain had a distinctive fruit aroma compared to other yeast strains exhibiting a fruit aroma.

Example 6: Production of chocolate using selected yeast strains Cocoa beans and cocoa pulp are fermented using two yeast strains whose strong fruit aroma has been confirmed by the selection method of Example 5, and chocolate is produced from the fermented product as a raw material. It was manufactured and sensory evaluated.

(1) Preparation of fermented cacao pod contents and production of chocolate A fermented cacao pod contents was prepared in the same manner as in Example 4 except that Kluyveromyces lactis JCM5219 strain and Debaryomyces hansenii JCM5912 strain were used as yeast strains. Chocolate was produced from fermented pod contents.

(2) Sensory evaluation The sensory evaluation was performed in the same manner as in Example 4. Compared to the group not inoculated with yeast, the chocolate made from the fermented cacao pod contents inoculated with each yeast strain may have the same flavor as that confirmed in the fermented cacao extract (Example 5). confirmed.

Claims (13)

A method for screening yeast that produces a fermented product having a desired flavor characteristic, which comprises culturing a test yeast in a culture medium containing a cocoa mass extract and saccharides, and is a screening method for the desired flavor. A method in which the properties are fruit scent, brewed liquor scent, distilled liquor scent or floral scent, and the saccharide contains at least glucose. The screening method according to claim 1, wherein the desired flavor characteristics are different from the flavor characteristics of the separation source of the test yeast. The test yeast is a yeast selected from the group consisting of yeasts of the genus Devaliomyces, yeasts of the genus Kluyberomyces, yeasts of the genus Saccharomyces, yeasts of the genus Zygosaccharomyces, yeasts of the genus Pikia, yeasts of the genus Candida and yeasts of the genus Hansenia spora. The screening method according to claim 1 or 2. The screening method according to any one of claims 1 to 3, wherein the concentration of the cocoa mass extract in the culture medium is 1 to 30% by mass (in terms of solid content). The screening method according to any one of claims 1 to 4, wherein the concentration of the added saccharide in the culture medium is 0.1 to 18% by mass. The screening method according to any one of claims 1 to 5 is carried out to select yeast that produces a fermented product having the desired flavor characteristics, and the selected yeast is added to the cacao raw material for fermentation. A method for producing a fermented product, which comprises. The production method according to claim 6, wherein the yeast belongs to Kluyveromyces lactis, Devaliomyces Hanseni, Kluyveromyces marcianus or Saccharomyces cerevisiae. The production method according to claim 6, wherein the cocoa raw material is selected from the group consisting of cocoa beans, cocoa pulp, and processed products thereof. The production method according to claim 8, wherein the processed product is cocoa pulp juice, cocoa mass and cocoa mass extract. The production method according to claim 6, wherein the cacao raw material contains cocoa beans and the fermented product is fermented cocoa beans. A method for producing cacao mass, which comprises carrying out the production method according to claim 10 to prepare fermented cacao beans, and preparing cacao mass using the prepared fermented cacao beans as a raw material. A method for producing cocoa or cocoa butter, which comprises preparing cocoa mass by carrying out the production method according to claim 11 and preparing cocoa or cocoa butter from the prepared cocoa mass. The production method according to claim 10 is carried out to prepare fermented cocoa beans, or the production method according to claim 11 is carried out to prepare cocoa mass, or the production method according to claim 12 is carried out. To prepare cocoa or cocoa butter, and to prepare an oily confectionery or cocoa-blended product using the prepared fermented cocoa beans, cocoa mass, cocoa or cocoa butter as a raw material. Production method.