KR20210078883A - Lactic acid bacterial fermentation products using fruits or vegetables, and manufacturing method thereof - Google Patents

Abstract

The present invention relates to a lactobacillus fermented food using an excellent strain and optimal fermentation conditions for the strain, a method for manufacturing the same, a granular food containing the strain, and a method for manufacturing the same. By the manufacturing method, a lactobacillus-containing fermented food healthful and easy to take in can be manufactured effectively. When the fermented food is manufactured, various fruit and vegetable are used, and thus new demand creation can be achieved and income can be created for fruit and vegetable farmers. In addition, the fermented food manufactured by the manufacturing method contains functional components in quantity, is easy to take in anytime and anywhere, and can be helpful for health promotion.

Description

Lactobacillus fermented food using fruits and vegetables and manufacturing method thereof {LACTIC ACID BACTERIAL FERMENTATION PRODUCTS USING FRUITS OR VEGETABLES, AND MANUFACTURING METHOD THEREOF}

The present invention relates to lactic acid bacteria fermented food using fruits and vegetables and a method for manufacturing the same.

There are many traditional fermented foods in Korea using dietary fiber and starch such as kimchi, pickled vegetables, soybean paste, gochujang, and makgeolli. These fermented plant foods have been recognized for their health functionality and superiority through modern science and technology because of the abundance of lactic acid bacteria, beneficial intestinal bacteria, and dietary fiber in plant foods.

The microbiome, the intestinal microbiome, affects the absorption and metabolism of nutrients, immunity, and the occurrence of various diseases in the intestinal tract, and plays a major role through interaction with the human body. Lactobacillus inhibits the growth of harmful bacteria by competition with harmful bacteria for nutrients in the intestine and secretion of antibacterial substances, prevents harmful bacteria from adhering to intestinal epithelial cells, and promotes the intestinal microbiome in a positive direction through effects such as inhibition of toxin production. They can play a big role as transformative probiotics.

In addition, dietary fiber contained in fermented foods is usually called prebiotics and can serve as food for beneficial intestinal bacteria, thereby helping the growth and settlement of probiotic lactic acid bacteria in the intestinal tract. Among traditional fermented foods, kimchi and doenjang, which are representative fermented foods rich in lactic acid bacteria and dietary fiber, are being studied for taste and functional substances.

Among fruits and vegetables, carrots are a food with about 500 kinds of phytochemicals, that is, natural plant ingredients. Nutritional and functional ingredients contained in carrots include beta-carotene, flavonoids, vitamins A, C, B1, B3, B5, K, potassium, magnesium, and folic acid. , high blood pressure prevention, anti-aging, and other health functionalities. However, according to a crop production survey by the National Statistical Office, domestic carrot production is declining from 118,594 tons in 2015 to 73,061 tons in 2016, 74.027 tons in 2017, and 73,143 tons in 2018.

Another functional vegetable, bok choy, is a food ingredient produced and used a lot in China. It is soft and does not have a special smell or taste, so there is less resistance to the bitter taste of vegetables with general chlorophyll, calcium, vitamins A, C, beta-carotene. It also contains a lot of nutrients and dietary fiber, so it has high value as a material for prebiotics and health functional foods. However, there are few studies on fermented foods using them.

Korean Patent Registration No. 10-0426279 (2004.04.08. Announcement)

In order to solve the above problems, an object of the present invention is to provide a lactic acid bacterium fermented food using an excellent strain and optimal fermentation conditions thereof, and a method for manufacturing the same.

In addition, an object of the present invention is to provide a granular food containing the strain and a method for producing the same.

The method for producing lactic acid bacteria fermented food according to the present invention comprises the steps of preparing a dry fruit and vegetable stock solution; adding water and a sugar source to the prepared stock solution; And Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus plantarum ( Lactobacillus plantarum ), or a mixed lactic acid bacteria thereof was inoculated into the medium and cultured, and then the cultured lactic acid bacteria cells were mixed with the stock solution to which the water and the sugar source were added. It may include; fermenting.

The lactic acid bacteria fermented food according to the present invention can be manufactured according to the above manufacturing method.

The method for producing a granular food containing lactic acid bacteria according to the present invention is a fruit and vegetable powder and Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) or Lactobacillus plantarum ( Lactobacillus plantarum ) Lactobacillus, or a lactic acid bacteria fermentation broth containing at least one of the above lactic acid bacteria is mixed preparing a stock solution; granulating the prepared fruit and vegetable stock solution; and drying the granulated fruit and vegetable stock solution.

The granular food containing lactic acid bacteria according to the present invention may be manufactured according to the above manufacturing method.

By the manufacturing method according to the present invention, it is possible to effectively produce a fermented food containing lactic acid bacteria that is beneficial to health and convenient to consume. By using a variety of fruits and vegetables, it can create new demand and help generate profits for farmers who were cultivating fruits and vegetables.

Fermented food and granular food prepared according to the above manufacturing method contain a large amount of functional ingredients, are easily distributed, and can be easily consumed anytime, anywhere, which can help promote health.

1 is a strain cultured according to a preparation example of the present invention.
2 is a result of measuring changes in absorbance (OD) and pH during fermentation at 25° C. of Lactobacillus fermentum (hereinafter, L. fermentum ) according to an experimental example of the present invention.
3 is a result of measuring the changes in glucose (glucose), lactate (lactate), acetate (acetate) and ethanol (ethanol) during 25 ℃ fermentation of L. fermentum.
Figure 4 is a result of measuring the change in OD and pH during 30 ℃ fermentation of L. fermentum.
5 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. fermentum at 30°C.
6 is a result of measuring the change in OD and pH during 37 ℃ fermentation of L. fermentum.
7 is a result of measuring the changes in glucose, lactate, acetate and ethanol during 37 ℃ fermentation of L. fermentum.
8 is a result of comparing the change according to the culture temperature during fermentation of L. fermentum , (A) is a graph comparing the change in OD and pH according to the culture temperature, (B) is glucose consumption according to the culture temperature and A graph comparing the production of lactate, (C) is a graph comparing the production of acetate and ethanol according to the culture temperature.
9 is a result of measuring changes in OD and pH during fermentation at 25° C. of Lactobacillus plantarum (hereinafter, L. plantarum ) according to an experimental example of the present invention.
10 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. plantarum at 25°C.
11 is a result of measuring the change in OD and pH during 30 ℃ fermentation of L. plantarum.
12 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. plantarum at 30°C.
13 is a result of measuring the change in OD and pH during 37 ℃ fermentation of L. plantarum.
14 is a result of measuring changes in glucose, lactate, acetate, and ethanol during 37° C. fermentation of L. plantarum.
15 is a result of comparing fermentation characteristics according to culture temperature during fermentation of L. plantarum , (A) is a graph comparing changes in OD and pH according to culture temperature, (B) is glucose consumption according to culture temperature A graph comparing the production of lactate and lactate, (C) is a graph comparing the production of acetate and ethanol according to the incubation temperature.
16 is a result of measuring changes in OD and pH during fermentation at 25° C. of Lactobacillus rhamnosus (hereinafter, L. rhamnosus ) according to an experimental example of the present invention.
17 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. rhamnosus at 25°C.
18 is a result of measuring changes in OD and pH during 30° C. fermentation of L. rhamnosus.
19 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. rhamnosus at 30°C.
20 is a result of measuring changes in OD and pH during 37 °C fermentation of L. rhamnosus.
21 is a result of measuring changes in glucose, lactate, acetate and ethanol during 37° C. fermentation of L. rhamnosus.
22 is a result of comparing the change according to the culture temperature during fermentation of L. rhamnosus , (A) is a graph comparing the change in OD and pH according to the culture temperature, (B) is the consumption of glucose according to the culture temperature and A graph comparing the production of lactate, (C) is a graph comparing the production of acetate and ethanol according to the culture temperature.
23 is a result of measuring changes in OD and pH during 25° C. fermentation of Leuconostoc mesenteroides (hereinafter, L. mesenteroides ) according to an experimental example of the present invention.
24 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. mesenteroides at 25°C.
25 is a result of measuring the change in OD and pH during 30 ℃ fermentation of L. mesenteroides.
26 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. mesenteroides at 30°C.
27 is a result of measuring the change in OD and pH during 37 ℃ fermentation of L. mesenteroides.
28 is a result of measuring changes in glucose, lactate, acetate, and ethanol during 37° C. fermentation of L. mesenteroides.
29 is a result of comparing the change according to the culture temperature during fermentation of L. mesenteroides , (A) is a graph comparing the change in OD and pH according to the culture temperature, (B) is glucose consumption according to the culture temperature and A graph comparing the production of lactate, (C) is a graph comparing the production of acetate and ethanol according to the culture temperature.
30 is a result of measuring changes in OD and pH during fermentation at 25° C. of Lactobacillus acidophilus (hereinafter, L. acidophilus ) according to an experimental example of the present invention.
31 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. acidophilus at 25°C.
32 is a result of measuring changes in OD and pH during fermentation of L. acidophilus at 30°C.
33 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. acidophilus at 30°C.
34 is a result of measuring changes in OD and pH during 37° C. fermentation of L. acidophilus.
35 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. acidophilus at 37°C.
Figure 36 is a result of comparing the change according to the culture temperature during fermentation of L. acidophilus , (A) is a graph comparing the change in OD and pH according to the culture temperature, (B) is glucose consumption according to the culture temperature and A graph comparing the production of lactate, (C) is a graph comparing the production of acetate and ethanol according to the culture temperature.
37 is a result of measuring changes in OD and pH during fermentation at 25° C. of Lactobacillus reuteri (hereinafter, L. reuteri ) according to an experimental example of the present invention.
38 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. reuteri at 25°C.
Figure 39 is a result of measuring the change in OD and pH during 30 ℃ fermentation of L. reuteri.
40 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. reuteri at 30°C.
41 is a result of measuring the change in OD and pH during 37 ℃ fermentation of L. reuteri.
42 is a result of measuring changes in glucose, lactate, acetate and ethanol during fermentation of L. reuteri at 37°C.
43 is a result of comparing the change according to the culture temperature during fermentation of L. reuteri , (A) is a graph comparing the change in OD and pH according to the culture temperature, (B) is glucose consumption according to the culture temperature and A graph comparing the production of lactate, (C) is a graph comparing the production of acetate and ethanol according to the culture temperature.
Figure 44 is a result of analyzing the heat resistance of each strain according to an experimental example of the present invention, (A) at 45 ℃, (B) at 55 ℃, (C) at 65 ℃ (heat shock) It is a graph in the case of giving
45 is a result of a mixture of dried carrots and dried bok choy in different ratios according to an embodiment of the present invention.
46 is a fermented beverage prepared according to Example 1 of the present invention.
47 is a graph analyzing the number of viable cells and changes in pH of a control stock solution according to an experimental example of the present invention.
48 is a graph showing changes in sugar component consumption and fermentation products of the control stock solution.
49 is a graph analyzing the number of viable cells and changes in pH of L. fermentum according to an experimental example of the present invention.
50 is a graph showing changes in sugar component consumption and fermentation products of L. fermentum.
51 is a graph analyzing the number of viable cells and changes in pH of L. plantarum according to an experimental example of the present invention.
52 is a graph showing changes in sugar component consumption and fermentation products of L. plantarum.
53 is a graph analyzing the number of viable cells and changes in pH of L. rhamnosus according to an experimental example of the present invention.
54 is a graph showing changes in sugar component consumption and fermentation products of L. rhamnosus.
55 is a graph analyzing the number of viable cells and changes in pH of L. mesenteroides according to an experimental example of the present invention.
56 is a graph showing changes in sugar component consumption and fermentation products of L. mesenteroides.
57 is a graph analyzing the number of viable cells and changes in pH of L. acidophilus according to an experimental example of the present invention.
58 is a graph showing changes in sugar component consumption and fermentation products of L. acidophilus.
59 is a graph analyzing the number of viable cells and changes in pH of L. reuteri according to an experimental example of the present invention.
60 is a graph showing changes in sugar component consumption and fermentation products of L. reuteri.
61 to 66 are results of evaluation of low-temperature storage properties of fermented beverages prepared using six types of bacteria.
67 is a fermented beverage prepared according to Example 2 of the present invention.
68 to 70 are results of analysis of fermentation characteristics according to the concentration of the sugar source, FIG. 68 shows 20 mL of purified water and 15 g of sugar source, FIG. 69 shows 10 mL of purified water and 25 g of sugar source, and FIG. 70 shows fermentation with the addition of 35 g of sugar source.
71 to 73 are results of analyzing the fermentation characteristics according to the culture temperature, and FIG. 71 shows fermentation at 25° C., FIG. 72 shows 30° C., and FIG. 73 shows fermentation at 37° C.
74 to 76 are results of analysis of fermentation characteristics according to the initial strain concentration, and FIG. 74 is 5 mL, FIG. 75 is 25 mL, and FIG. 76 is fermented at a concentration of 50 mL.
77 is an image of the granule product prepared according to Example 3 of the present invention.
78 is a storage evaluation result of lactic acid bacteria-containing carrots and bok choy granulated products prepared according to Example 3 of the present invention.

Hereinafter, the present invention will be described in detail.

The present inventor tried to establish the optimal mixing ratio and fermentation conditions by using the probiotic lactic acid bacteria strain approved for use by the Food and Drug Administration, and by exploring the appropriate fermentation conditions according to the contents of carrots, bok choy, plums, and peaches. In addition, in order to diversify products, the present invention was completed by trying to produce a powder product of carrot and bok choy lactic acid bacteria containing lactic acid bacteria or a fermented solution thereof.

The present invention provides a step of preparing a dry fruit and vegetable stock solution, adding water and a sugar source to the prepared stock solution, and Lactobacillus rhamnosus , Lactobacillus plantarum ), or a mixed lactic acid bacteria thereof in a medium After inoculation and culturing, it provides a method for producing a fermented lactic acid bacteria food comprising the step of fermenting by mixing the cultured lactic acid bacteria cells in the stock solution added with the water and sugar source.

In the method for producing lactic acid bacteria fermented food according to the present invention, the step of preparing the dried fruit and vegetable stock solution is to put the dried fruit and vegetable in water and pulverize it, then at 110 to 130° C., preferably at 120 to 125° C., for 10 to 30 minutes. , preferably by autoclaving for 20 minutes.

The dried fruits and vegetables may include, but are not limited to, carrots, bok choy, avocados, kale, celery, beets, or a mixture thereof.

Preferably, the dried fruits and vegetables may include dried carrots and dried bok choy, and the dried carrots and dried bok choy may be included in a weight ratio of (5 to 15): 1, more preferably 10: 1. According to an embodiment of the present invention, it was confirmed that the color and the preference when the weight ratio is suitable for manufacturing a fermented beverage.

The dried carrots and dried bok choy can be prepared by cultivating or purchasing carrots and bok choy, or dried carrots and dried bok choy.

Fruits and vegetables, which are the main raw materials of the fermented lactic acid bacteria food according to the present invention, have the advantage of easy storage and distribution because dried fruits and vegetables are used.

In the method for producing a fermented lactic acid bacteria food according to the present invention, after the dried fruit and vegetable stock solution is prepared, the step of adding water and a sugar source to the prepared stock solution may be performed. The water and sugar source may be added in 1/3 to 1/5 of the weight of the dried fruit and vegetable stock solution.

The sugar source may include, but is not limited to, juice or concentrate of fruits such as plums, peaches, apples, grapes, pears, and peaches. By selecting and manufacturing a sugar source using the fruit, it is possible to realize a healthy and diverse taste and flavor without added sugar.

Preferably, the sugar source may include peach or plum juice concentrate, and the water and the sugar source are mixed in a weight ratio of 1: (1 to 4), more preferably 1: (2 to 3) by weight. It may be added to the stock solution. When water and sugar source in the above range are added to the stock solution, it is possible to promote the fermentation of the lactic acid bacteria, and the taste and aroma are also excellent.

In the method for producing lactic acid fermented food according to the invention, the step of the fermentation, Lactobacillus ramno suspension (Lactobacillus rhamnosus), Lactobacillus Planta column (Lactobacillus plantarum), or a mixture of lactic acid bacteria, preferably Lactobacillus ramno suspension (Lactobacillus rhamnosus ) after inoculation and culturing in the medium, it can be carried out by mixing the cultured lactic acid bacteria cells in the stock solution to which the water and the sugar source are added.

The lactic acid bacteria can be fermented by pre-culturing and using the culture solution or by centrifuging to remove only the cells from which the supernatant of the culture solution is removed. Preferably, the lactic acid bacteria are inoculated into 20 to 50 mL of MRS medium, more preferably 25 mL, and then centrifuged, and the lactic acid bacteria cells obtained by centrifugation are mixed with the stock solution to which the water and the sugar source are added, and 30 to 40 ° C. In, more preferably, it can be fermented at 37 ℃. When fermented in the above range of culture or fermentation conditions, it is possible to efficiently manufacture fermented food by increasing productivity.

The present invention provides a lactic acid bacteria fermented food prepared according to the method for producing the lactic acid bacteria fermented food.

The fermented food may include beverages, dairy products, and processed foods such as various drinks, canned food, and jam, and may preferably be lactic acid bacteria fermented fruit and vegetable beverages, but is not limited thereto, and may be applied to various foods.

In addition, the present invention comprises the steps of preparing a fruit and vegetable stock solution by mixing a vegetable powder and Lactobacillus rhamnosus or Lactobacillus plantarum lactic acid bacteria, or a lactic acid bacteria fermentation broth containing one or more of the above lactic acid bacteria; It provides a method for producing a granular food containing lactic acid bacteria, comprising the steps of granulating the prepared fruit and vegetable stock solution and drying the granulated fruit and vegetable stock solution.

In the method for producing a granular food containing lactic acid bacteria according to the present invention, the step of preparing the raw vegetable and vegetable solution comprises one or more lactic acid bacteria of fruit and vegetable powder and Lactobacillus rhamnosus or Lactobacillus plantarum, or the lactic acid bacteria. Lactobacillus fermentation broth containing one or more, preferably Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) Or it can be carried out by mixing its fermentation broth.

The fruit and vegetable powder may be included in an amount of 15 to 30 parts by weight based on 100 parts by weight of the total food, and the fruit and vegetable powder and the lactic acid bacteria or lactic acid bacteria fermentation broth may be mixed in a weight ratio of (2 to 4): 1. The above range is preferable because the fruit and vegetable stock solution must be prepared within the above range so that granules can be well formed in the subsequent granulation step.

The fruit and vegetable powder may be a powder of one or more fruits and vegetables selected from the group consisting of carrots, bok choy, avocado, kale, celery and beets, but is not limited thereto. Preferably, it may be carrot powder, bok choy powder, or a mixed powder thereof.

The fruit and vegetable powder may be prepared by cultivating or purchasing the fruits and vegetables, drying them, and then pulverizing them, or may be purchased in the form of fruits and vegetables powder.

The vegetable stock solution may further include one or more selected from the group consisting of lactose, dextrin, glucose, corn starch, refined salt, DHA, powdered sugar, and nucleic acid IG, but is not limited thereto.

In the method for producing a granular food containing lactic acid bacteria according to the present invention, the granulating of the prepared fruit and vegetable stock solution may be granulated using an extrusion granulator, but is not limited thereto.

In the method for producing a granulated food containing lactic acid bacteria according to the present invention, the drying of the granulated fruit and vegetable stock solution is 60% so that the water content of the granulated fruit and vegetable stock solution is 2 to 4 wt%, preferably 2.5 to 3.5%. to 70°C, preferably at 65°C.

In the method for manufacturing a granular food containing lactic acid bacteria according to the present invention, the manufacturing method may further include the steps of selecting the dried granules using a vibrating sieve after the drying and packaging the selected granules. can

The present invention provides a lactic acid bacteria-containing granular food prepared according to the method for manufacturing a granular food containing lactic acid bacteria. The food may be prepared in various forms, such as snacks, processed food, food additives, or may be prepared in a form contained in food in a conventional sense. Since the food is manufactured using fruits and vegetables, it can have a synbiotic effect and thus can be prepared as food to which various types of health functionalities are imparted.

Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the contents of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

<Preparation Example 1> Strain selection

Six types of lactic acid bacteria known as probiotics were sold from the Korea Agricultural and Microbiology Resource Center (KACC). MRS broth and bacto agar (Difco, MI, USA) were used as the medium for lactic acid bacteria growth.

6 kinds of lactic acid bacteria sold from KACC, Lactobacillus fermentum KACC11441, Lactobacillus plantarum KACC11451, Lactobacillus rhamnosus KACC11953, Leuconostok mesenteroides KACC12312 ), Lactobacillus acidophilus (Lactobacillus acidophilus KACC12419), and Lactobacillus reuteri (Lactobacillus reuteri KACC11452) ampoules were cut using an ampoule cutter. After cutting, the cotton plug in the ampoule was removed and flame sterilized. Thereafter, the cells were suspended using sterile physiological saline, and then inoculated into MRS medium and MRS agar medium at the same time. A single colony appeared in each sample medium was selected, separated and cultured in MRS liquid medium.

1 is a strain cultured according to a preparation example of the present invention, A is Lactobacillus fermentum ( Lactobacillus fermentum ), B is Lactobacillus plantarum ( Lactobacillus plantarum ), C is Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), D is Leuconostoc mesenteroides ( Leuconostoc mesenteroides ), E is Lactobacillus acidophilus ( Lactobacillus acidophilus ), F is Lactobacillus reuteri ( Lactobacillus reuteri ).

<Experimental Example 1> Analysis of strain characteristics

An experiment was conducted to select the bacteria acquired from KACC for the purpose of application to fermented drinks of carrots and bok choy. First, in order to examine the effect of temperature on each strain, fermentation was performed under three conditions of 25°C, 30°C, and 37°C, and absorbance of 600 nm and pH were measured. Analysis of fermentation products was also used. Based on the experimental results, selection criteria for strains to be used for fermented beverages were set.

1. Lactobacillus fermentation method

For fermentation, 5mL of MRS medium was dispensed into test tubes and 1% of the KACC strain stored at -80°C was inoculated and cultured overnight at 80rpm in a shaking incubator (VS-8480, Vision Scientific, Bucheon, Korea). The culture was inoculated at a concentration of 1% in 50 mL of MRS liquid medium dispensed in a 250 mL Erlenmeyer flask, and fermented for 48 hours at 80 rpm in a shaking incubator at 25°C, 30°C, and 37°C.

2. Physicochemical characterization

1) Cell growth

Absorbance (Optical density, OD) was measured to observe the cell growth of each strain. Absorbance was measured at 600 nm using a UV-1800 spectrophotometer (Shimadzu Inc., Kyoto, Japan).

2) pH

In order to observe the change in pH in the medium during fermentation of each strain, a 1 mL sample of the fermentation broth was taken and the pH was measured using a Beckman 390 pH meter (Beckman Coulter Inc., CA, USA).

3) HPLC

For quantitative analysis of the fermentation product of each strain, a sample of 1 mL of fermentation broth was taken, centrifuged at 13,500 rpm for 10 minutes, and 700 μL of the supernatant was filtered with a 0.2 μm Nalgene syringe filter (Thermo Scientific Inc., MA, USA), followed by HPLC. was analyzed with For HPLC, an Agilent 1260 Infinity Ⅱ (Agilent, Santa Clara CA, USA) was used, and the column was an Aminex HPX-87H Column (300mm×7.5mm, 9μm, Bio-Rad, CA, USA), and the mobile phase was solvent 0.005. M sulfuric acid was used. The temperature of the column oven was 60° C., the sample injection amount was 10 μL, and the flow rate was 0.6 mL/min.

3. Analysis Results

3-1. Lactobacillus Fermentum ( Lactobacillus fermentum ; Below, L. fermentum ) cell growth, pH, fermentation product analysis

1) 25℃

Referring to FIG. 2 , the OD value of L. fermentum fermentation at 25° C. reached a maximum of 7.06 at 36 hours after fermentation and decreased to 6.46 after 48 hours of fermentation. The pH value reached the lowest value of 4.48 at 36 hours, and the pH value gradually increased after 36 hours of fermentation, and the pH value increased to 4.52 after 48 hours of fermentation.

Referring to FIG. 3 , glucose was analyzed as 0 g/L at 36 hours, and 7.3 g/L was consumed between 12 hours and 24 hours during the time when glucose was most consumed. Lactate continued to increase until 48 hours and was finally analyzed to be 9.7 g/L. Acetate (acetate) showed only an increase from 3.8 g/L at the beginning of fermentation to 4.3 g/L after 48 hours of fermentation, and ethanol (ethanol) produced 4.2 g/L during fermentation for 48 hours.

2) 30℃

Referring to FIG. 4 , the OD value of L. fermentum fermentation at 30° C. reached a maximum of 7.14 at 24 hours and decreased to 6.24 after 48 hours of fermentation. The pH value reached the lowest value of 4.44 at 24 hours, and after 24 hours of fermentation, the pH value increased and rose to 4.50 after 48 hours of fermentation.

Referring to FIG. 5 , glucose was analyzed as 0 g/L at 24 hours, and 8.3 g/L was consumed between 12 and 24 hours during the time when glucose was most consumed. Lactate produced 9.9 g/L at 36 hours, reaching the maximum lactate level, and greatly increased at the beginning of fermentation. Acetate increased from 3.9 g/L to 4.3 g/L during 48 hours of fermentation and ethanol was analyzed to be 4.1 g/L after 48 hours of fermentation.

3) 37℃

Referring to FIG. 6 , the OD value of L. fermentum fermentation at 37° C. reached a maximum of 6.41 at 12 hours and decreased to 4.8 after 48 hours of fermentation. The pH value reached the lowest value of 4.44 at 12 hours, and the pH value increased after 12 hours and rose to 4.5 after 48 hours of fermentation.

Referring to FIG. 7 , glucose was analyzed as 0 g/L at 12 hours, and 15.3 g/L was consumed between 6 hours and 12 hours when glucose was most consumed. Lactate reached the maximum value of 9.7 g/L at 36 hours and significantly increased in the initial stage of fermentation. Acetate increased from 3.9 g/L at the beginning of fermentation to 4.3 g/L after 48 hours of fermentation. Ethanol was measured at 4 g/L through 48 hours of fermentation.

4) L. fermentum change with the incubation temperature of

Referring to Figure 8, when comparing the fermentation at 25 ℃, 30 ℃, 37 ℃ of L. fermentum , the growth rate of lactic acid bacteria was found to be faster as it increased from 25 ℃ to 37 ℃. However, unlike the growth rate of lactic acid bacteria, the maximum growth value of lactic acid bacteria was not proportional to the increase in temperature. Compared to the OD peaks of 25°C and 30°C fermentations, the highest OD values of the 37°C fermentation were lower. Through this, it can be confirmed that L. fermentum increases the growth rate of bacteria as the temperature increases, but the growth of bacteria may be less when fermented at a temperature higher than 30° C. compared to other temperatures.

When the pH graph was compared, the time to reach the minimum value for each temperature was found to be faster as the temperature increased, and the pH graph for each temperature showed less change in the minimum value according to the temperature difference. When the OD graph and the pH graph are compared, it can be seen that the pH value is at the lowest value when the OD value reaches the highest value.

Comparing the glucose graphs according to the temperature change, it was found that the glucose was consumed faster as the temperature increased. Comparing the lactate graphs, it can be seen that although the rate of lactate formation gradually increases as the temperature increases, there is no significant difference in the amount of lactate finally produced even when the temperature is changed. In addition, it was found that a large amount of lactate was produced at a time when glucose was decreased, and there was no change in the amount of lactate when all of the glucose was consumed.

There was no difference in the production of acetate according to temperature change, and in the case of ethanol, the rate of ethanol production increased as the temperature increased, but there was no significant change in the amount of ethanol finally produced after 48 hours of fermentation.

Through this, it can be confirmed that L. fermentum increases the consumption rate of sugar and the production rate of metabolites as the temperature increases, but the effect on the final production of metabolites by temperature change is small. The growth of lactic acid bacteria also increases the growth rate as the temperature increases, but it can be seen that the maximum amount of lactic acid bacteria is measured at 25 ℃ and 30 ℃ higher than 37 ℃.

3-2. Lactobacillus plantarum ( Lactobacillus plantarum ; Below, L. plantarum ) cell growth, pH, fermentation product analysis

1) 25℃

Referring to FIG. 9 , the OD value of L. plantarum fermentation at 25° C. reached a maximum of 13.56 at 36 hours, and gradually decreased after 36 hours to 13.08 at 48 hours. The pH value reached the lowest value of 4.01 at 30 hours, and after 30 hours, the pH value increased and rose to 4.19 at 48 hours after fermentation.

Referring to FIG. 10 , glucose was analyzed as 0 g/L at 36 hours, and 9.9 g/L was consumed between 6 hours and 12 hours when glucose was most consumed. Lactate produced 19 g/L at 36 hours, reaching the maximum lactate level, and acetate increased from 3.6 g/L at the beginning of fermentation to 5 g/L after 48 hours of fermentation. And no ethanol was produced during the 48 hour fermentation.

2) 30℃

Referring to FIG. 11 , the OD value of L. plantarum fermentation at 30° C. reached a maximum value of 11.96 at 18 hours and gradually decreased after 18 hours to decrease to 10.12 at 48 hours. The pH value reached the lowest value of 3.94 at 24 hours, and after 24 hours, the pH value increased and rose to 4.01 after 48 hours of fermentation.

Referring to FIG. 12 , glucose was all consumed in 24 hours, and 10.4 g/L was consumed for 6 hours between 6 hours and 12 hours when the consumption of glucose was the fastest. Lactate reached a maximum at 36 hours of fermentation, and acetate increased from 3.7 g/L at the beginning of fermentation to 4.7 g/L after 48 hours of fermentation. Also, no ethanol was produced during the 48 hour fermentation.

3) 37℃

Referring to FIG. 13 , the OD value of L. plantarum fermentation at 37° C. reached a maximum value of 8.1 at 18 hours and gradually decreased after 18 hours to decrease to 6.1 at 48 hours. The pH value reached the lowest value of 4.02 at 36 hours.

Referring to FIG. 14 , glucose was all consumed in 24 hours, and 13.5 g/L was consumed between 6 hours and 12 hours when the consumption of glucose was the highest. Lactate continued to increase until 48 hours to produce 17.5 g/L, reaching the maximum lactate level. Acetate increased from 3.7 g/L at the beginning of fermentation to 4.7 g/L after 48 h fermentation, and ethanol was not produced during 48 h fermentation.

4) L. plantarum change with the incubation temperature of

Referring to Figure 15, when comparing the fermentation at 25 ℃, 30 ℃, 37 ℃ of L. plantarum , the rate of reaching the maximum of lactic acid bacteria growth was found to be faster as it increased from 25 ℃ to 37 ℃. However, unlike the time to reach the maximum value, the maximum growth value of lactic acid bacteria did not increase as the temperature increased, and the maximum growth of lactic acid bacteria was achieved at a low temperature. Through this, it can be confirmed that L. plantarum can obtain more lactic acid bacteria when fermentation is performed at a low temperature, although the time for the bacteria to reach the maximum value increases as the temperature increases.

When the pH graphs were compared, the pH graphs at 30°C and 37°C had similar values, but unlike the two temperatures, the pH graph at 25°C showed that the pH value decreased slowly during the first 12 hours. The pH value of 25 ℃ had similar values to the pH values of 30 ℃ and 37 ℃ after 12 hours, but gradually increased after 36 hours, indicating a relatively higher pH value than other temperatures after 48 hours of fermentation.

When the glucose graphs were compared, it was found that the rate of glucose consumption was slow in fermentation at 25°C compared to 30°C and 37°C. Through this, L. plantarum can be confirmed that the higher the temperature, the faster the consumption rate of glucose. In addition, from the fact that the OD graphs at 30°C and 37°C reached their maximum values at 18 hours, it can be predicted that glucose was all consumed within 18 hours in the fermentation at 30°C and 37°C.

Comparing the lactate production graph, the result of fermentation at 37°C had a similar value to that of fermentation at 30°C up to 12 hours, but showed a difference from 24 hours, showing a lower value than the lactate production at 30°C. , at 25°C, lactate was produced slowly compared to the other two temperatures. Through this, it can be determined that it is appropriate to proceed with fermentation at 30° C. in order to obtain the greatest amount of lactate. Acetate was increased in fermentations at 25°C and 30°C except for 37°C, and in the case of ethanol, it was not produced at all three temperatures.

In conclusion, L. plantarum can obtain the most lactic acid bacteria when fermentation is carried out at 25℃, but it can be confirmed that fermentation takes longer than when fermentation is performed at 30℃.

3-3. Lactobacillus rhamnosus ( Lactobacillus rhamnosus ; Below, L. rhamnosus ) cell growth, pH, fermentation product analysis

1) 25℃

Referring to FIG. 16 , the OD value of L. rhamnosus fermentation at 25° C. reached the maximum value of 12.6 at 30 hours, and gradually decreased after 30 hours after fermentation, and decreased to 11.66 at 48 hours. Also, the pH value reached the lowest value of 4.19 at 30 hours.

Referring to FIG. 17 , glucose was all consumed at 36 hours, and the fastest period of glucose consumption was between 12 and 24 hours, consuming a total of 8 g/L. Lactate produced 15 g/L during 48 hours of fermentation, and acetate increased from 3.7 g/L at the beginning of fermentation to 4 g/L after 48 hours of fermentation. No ethanol was produced during the 48 hour fermentation.

2) 30℃

Referring to FIG. 18 , the OD value of L. rhamnosus fermentation at 30° C. reached the maximum value of 12.86 at 30 hours and gradually decreased after 30 hours to 11.86 at 48 hours. The pH value reached a minimum of 4.14 at 36 hours.

Referring to FIG. 19 , glucose was measured to be 0 g/L at 24 hours, and 8.7 g/L was consumed between 12 and 24 hours during the time when glucose was most consumed. Lactate reached a maximum of 14.5 g/L at 24 hours, and then slowly decreased to 13.6 g/L after 48 hours of fermentation. Acetate increased from 3.7 g/L at the beginning of fermentation to 4.6 g/L after 48 hours of fermentation, and ethanol was not produced during 48 hours of fermentation.

3) 37℃

Referring to FIG. 20 , the OD value of L. rhamnosus fermentation at 37° C. reached the maximum value of 11.14 at 18 hours of fermentation, and gradually decreased after 18 hours of fermentation, and decreased to 10.58 after 48 hours of fermentation. The pH value reached the lowest value of 4.09 at 18 hours, and gradually increased after 18 hours to 4.14 after 48 hours of fermentation.

Referring to FIG. 21 , glucose decreased to 0 g/L at 24 hours, and 12.3 g/L was consumed between 6 and 12 hours during the time when glucose consumption was the highest. Lactate reached a maximum of 16.1 g/L at 24 hours, and then slowly decreased to 15.3 g/L after 48 hours of fermentation. Acetate increased from 3.7 g/L at the beginning of fermentation to 4.6 g/L after fermentation for 48 hours. No ethanol was produced during the 48 hour fermentation.

4) L. rhamnosus change with the incubation temperature of

Referring to Figure 22, the rate of reaching the maximum value of lactic acid bacteria growth in the fermentation of L. rhamnosus at 25 ℃, 30 ℃, 37 ℃ was found to be faster as the temperature increased from 25 ℃ to 37 ℃. However, unlike the time to reach the maximum value, the maximum growth value of lactic acid bacteria did not increase as the temperature increased. The highest OD of the 37℃ fermentation is 11.14, the highest OD of the 30℃ fermentation is 12.86, and the highest OD of the 37℃ fermentation is 12.6, confirming that the highest growth of lactic acid bacteria is achieved at 30℃.

In addition, when the pH graphs were compared, it was found that the pH minimum was reached at a faster rate when fermented at a higher temperature. Since the decrease in pH is closely related to the production of lactate, it can be seen that the tendency to produce lactate also increases at a faster rate as the fermentation is carried out at a high temperature.

When the glucose graph was compared, it was found that the rate of glucose consumption was faster in the fermentation at 30°C and at 37°C compared to the fermentation at 25°C. Through this, it can be confirmed that L. rhamnosus increases the rate of glucose consumption when the temperature is high compared to the low temperature. In addition, through the 37 ℃ OD graph reached the maximum value after 18 hours of fermentation, it can be determined that glucose was all consumed around 18 hours in the 37 ℃ fermentation.

Comparing the lactate graphs, it was found that the 37°C lactate graph reached the highest value in the fastest time, and the highest value was also the highest. Also, in the lactate graphs at 30° C. and 37° C., it was found that the amount of lactate decreased after all of the glucose was consumed. From this, it can be confirmed that in order to obtain the greatest amount of lactate, fermentation must be finished when all of the glucose is consumed.

Acetate was less produced at 25 °C than at 30 °C and 37 °C, and ethanol was not produced at either 25 °C, 30 °C, or 37 °C.

In conclusion, in L. rhamnosus , the consumption of glucose and the production rate and production of lactate increased as the temperature increased. The growth rate of lactic acid bacteria was also rapid at high temperature, but the maximum growth rate of lactic acid bacteria was 30 compared to 37℃. It was measured to be high at ℃ and 25℃. Through this, it can be confirmed that it is appropriate to proceed with the fermentation at 37° C. in order to obtain more lactate, and it is preferable to proceed with the fermentation at 30° C. to obtain more lactic acid bacteria cells.

3-4. Leukonostok mecenteroides ( Leuconostoc mesenteroides ; Below, L. mesenteroides ) cell growth, pH, fermentation product analysis

1) 25℃

Referring to FIG. 23 , the OD value of L. mesenteroides fermentation at 25° C. reached a maximum of 4.61 at 30 hours and gradually decreased after 30 hours to decrease to 4.52 after 48 hours of fermentation. The pH value reached a minimum of 4.40 at 30 hours.

Referring to FIG. 24 , glucose was measured to be 0 g/L at 36 hours, and lactate was measured to be 10 g/L after 48 hours of fermentation. Acetate increased from 4 g/L at the beginning of fermentation to 5.8 g/L after 48 hours of fermentation, and ethanol produced 3.3 g/L after 48 hours of fermentation.

2) 30℃

Referring to FIG. 25 , the OD value of L. mesenteroides fermentation at 30° C. reached a maximum of 4.69 at 36 hours and gradually decreased to 4.52 after 48 hours of fermentation. After reaching the lowest value of 4.39 at 24 hours, the pH value gradually increased and increased to 4.47 after 48 hours of fermentation.

Referring to FIG. 26 , glucose was measured at 0 g/L at 24 hours, and lactate produced 9.4 g/L after 48 hours of fermentation. Acetate increased from 3.9 g/L at the beginning of fermentation to 5.9 g/L after 48 hours of fermentation, and ethanol produced 2.8 g/L after 48 hours of fermentation.

3) 37℃

Referring to FIG. 27 , the OD value of L. mesenteroides fermentation at 37° C. reached a maximum of 2.83 at 36 hours and gradually decreased to 2.58 after 48 hours of fermentation. The pH value reached the lowest value of 4.79 at 24 hours and did not change significantly until 48 hours.

Referring to FIG. 28 , the glucose of L. mesenteroides fermentation at 37° C. was not consumed for 48 hours, and 5.7 g/L of glucose remained. And lactate produced 5.8 g/L during 48 hours of fermentation. Acetate increased from 3.9 g/L at the beginning of fermentation to 5.2 g/L after 48 hours of fermentation, and ethanol produced 1.7 g/L after 48 hours of fermentation.

4) L. mesenteroides change with the incubation temperature of

Referring to Figure 29, when comparing the fermentation of L. mesenteroides at 25 ℃, 30 ℃, 37 ℃, the speed of reaching the maximum cell growth of lactic acid bacteria showed the fastest appearance at 30 ℃. At 25°C and 37°C, it was found to grow later than 30°C, and in particular, when fermentation was performed at 37°C, the cell growth was the lowest compared to other temperatures. In the case of pH, as in the OD graph, the pH decreased the least when fermented at 37° C. compared to the other two temperatures.

When the glucose graphs were compared, it was found that the fermentation at 30°C had a faster rate of glucose consumption compared to the fermentation at 25°C. It can be seen that the consumption value of glucose after 12 hours is sharply decreased compared to the previous consumption value.

Comparing the acetate graph, it can be seen that the acetate reached the maximum value faster at 30°C compared to other temperatures, and had a lower value than other temperatures at 37°C after 48 hours of fermentation. Ethanol also produced a lower amount at 37 °C compared to other temperatures, and 25 °C produced a slightly higher amount of ethanol than 30 °C.

In L. mesenteroides , when fermentation was carried out at 37°C, it was found that the growth was not completed completely and the fermentation was terminated compared to the fermentation carried out at other temperatures. Through this, it can be confirmed that L. mesenteroides has decreased cell activity at 37°C.

It can be seen that the rate of glucose consumption decreased after 12 hours during fermentation at 25°C and 30°C, which can be judged because the pH of L. mesenteroides reached a pH of 4.5 where cell activity was low. Also, since acetate and ethanol are produced together with lactate, it can be confirmed that the strain is performing heterolactic fermentation.

3-5. Lactobacillus acidophilus ( Lactobacillus acidophilus ; Below, L. acidophilus ) cell growth, pH, fermentation product analysis

1) 25℃

Referring to FIG. 30 , the OD value of L. acidophilus fermentation at 25° C. continued to increase until 48 hours to reach 1.14, and the pH value also continued to decrease to reach 5.21 at 48 hours.

Referring to FIG. 31 , glucose was not consumed during 48 hours of fermentation, leaving 13.2 g/L of glucose. Lactate produced 3.5 g/L during 48 hours of fermentation, and no acetate and ethanol were produced.

2) 30℃

Referring to FIG. 32 , the OD value of L. acidophilus 30° C. fermentation continued to increase until 48 hours to reach 6.08, and the pH value also continued to decrease to reach 4.24 at 48 hours.

Referring to FIG. 33 , glucose was not consumed during the 48 hours of fermentation, leaving 6.2 g/L of glucose. Lactate produced 11.8 g/L during 48 hours of fermentation and acetate increased from 3.7 g/L at the beginning of fermentation to 4.2 g/L after 48 hours of fermentation. And no ethanol was produced.

3) 37℃

Referring to FIG. 34 , the OD value of L. acidophilus 37° C. fermentation reached a maximum of 7.08 at 30 hours, gradually decreased after 30 hours of fermentation, and decreased to 6.08 at 48 hours after fermentation. The pH value continued to decrease for 48 hours and reached 3.958.

Referring to FIG. 35 , glucose reached 0 g/L at 48 h, and lactate produced 17.4 g/L during 48 h fermentation. Acetate increased from 3.7 g/L at the beginning of fermentation to 4.5 g/L after 48 hours of fermentation, and ethanol was not produced.

4) L. acidophilus change with the incubation temperature of

Referring to Figure 36, when comparing the fermentation at 25 ℃, 30 ℃, 37 ℃ of L. acidophilus , the speed to reach the maximum value of lactic acid bacteria growth was the fastest at 37 ℃, specifically, at 25 ℃ lactic acid bacteria Almost no growth was observed. When the pH graph was compared, the decrease in pH at 25°C was the smallest, and the decrease in pH at 30°C followed by a decrease in pH at 37°C.

When the glucose graphs were compared, it was found that the glucose consumption and consumption rate increased as the temperature increased. During fermentation at 25°C, almost no glucose was consumed, but during fermentation at 30°C, the consumption of glucose increased compared to fermentation at 25°C, but not all was consumed, and all glucose was consumed only at 37°C. Through this, it can be confirmed that L. acidophilus shows better consumption of sugar at high temperature than at low temperature. Lactate produced the most lactate at 37°C, where the consumption of glucose was the highest, and the lowest at 25°C, where the consumption of glucose was the lowest.

Comparing the acetate graph, the most acetate was produced at 37°C compared to other temperatures, but the total production was found to be insignificant at 1 g/L or less. Ethanol was not produced at all temperatures.

L. acidophilus showed almost no cell growth at 25℃, and it can be seen that the cell growth rate, glucose consumption rate, and lactate production rate increase as the temperature increases. Through this, it can be confirmed that L. acidophilus is a bacterium that grows better at a high temperature of 30℃ or higher.

3-6. Lactobacillus reuteri ( Lactobacillus reuteri ; Below, L. reuteri ) cell growth, pH, fermentation product analysis

1) 25℃

Referring to FIG. 37 , the OD value of L. reuteri fermentation at 25° C. reached a maximum of 5.26 at 36 hours, and gradually decreased after 36 hours to decrease to 5.17. The pH value showed little change until 18 hours, and started to decrease significantly after 18 hours, and decreased to 4.51 after 48 hours of fermentation.

Referring to FIG. 38 , glucose was all consumed at 48 hours, and glucose consumption was the most at 6.8 g/L from 24 hours to 36 hours. Lactate produced 8.4 g/L after 48 hours of fermentation. Acetate increased from 4 g/L at the beginning of fermentation to 5.7 g/L after 48 hours of fermentation, and ethanol produced 3 g/L.

2) 30℃

Referring to FIG. 39 , the OD value of L. reuteri fermentation at 30° C. reached a maximum of 9.46 at 30 hours and then gradually decreased to 8.92 after 48 hours of fermentation. The pH value decreased to the lowest value of 4.48 at 30 hours, increased little by little after 30 hours of fermentation, and increased to 4.6 after 48 hours of fermentation.

Referring to FIG. 40 , glucose was all consumed at 24 hours, and 10.9 g/L was consumed from 12 hours to 24 hours during the most consumed time of glucose. Lactate reached a maximum of 8.2 g/L at 24 hours and gradually decreased to 7 g/L after 48 hours of fermentation. Acetate increased from 3.9 g/L at the beginning of fermentation to 6.3 g/L after 48 hours of fermentation, and ethanol reached a maximum of 3 g/L at 24 hours from 0.3 g/L at the beginning of fermentation and gradually decreased after 48 hours of fermentation. was 2.8 g/L.

3) 37℃

Referring to FIG. 41 , the OD value of L. reuteri at 37° C. fermentation reached a maximum of 7.08 at 30 hours, and then gradually decreased to 6.08 after 48 hours of fermentation. The pH value decreased to the lowest value of 4.47 at 30 hours, and then gradually increased to 4.57 after 48 hours of fermentation.

Referring to FIG. 42 , glucose was all consumed at 12 hours, and the consumption of glucose from 12 hours to 24 hours was the most at 11.5 g/L. Lactate reached a maximum of 9 g/L at 12 hours and gradually decreased to 8.8 g/L after 48 hours of fermentation. Acetate increased from 3.9 g/L at the beginning of fermentation to 4.9 g/L after 48 hours of fermentation, and ethanol reached a maximum of 3.8 g/L at 12 hours from 0.2 g/L at the beginning of fermentation and gradually decreased at 48 hours. was 3.7 g/L.

4) L. reuteri change with the incubation temperature of

Referring to Figure 43, when comparing the fermentation at 25 ℃, 30 ℃, 37 ℃ of L. reuteri, the growth rate of lactic acid bacteria showed an increase as the temperature increased, but the growth of lactic acid bacteria was the highest at 30 ℃, , and the next highest temperature was 37°C. In addition, the growth of lactic acid bacteria did not appear until 18 hours at 25 ℃, and the growth of lactic acid bacteria was also the least.

When the pH graphs were compared, the first temperature decreasing to the minimum value was 37°C, and then the decreasing temperature was 30°C. The pH at 25° C. showed little change until 18 hours as shown in the OD graph, and then started to decrease after 18 hours, and finally reached pH 4.5 at all three temperatures.

When the glucose graphs were compared, it was found that the time to consume all the glucose increased as the temperature increased. At 25°C, all glucose was consumed in 48 hours, but at 30°C, all glucose was consumed in 24 hours, and at 37°C, all glucose was consumed in 12 hours. Through this, it can be confirmed that L. reuteri consumes more sugar at a higher temperature than at a lower temperature.

When the lactate graphs were compared, the production of lactate at 37° C. was the highest and the fastest. In the case of 30°C, the maximum value was reached at 24 hours, but the lactate decreased over time, and the production of lactate increased the latest at 25°C.

Comparing the acetate graph, it could be seen that the most acetate was produced at 30 ° C compared to other temperatures, and in many cases it was increased to 2 g/L. In the case of ethanol, it reached the maximum value (4 g/L) the fastest at 37°C, and produced the largest amount among the three temperatures. At 30°C, the peak was produced at 24 hours, but gradually decreased over time, and at 25°C, it increased slowly and reached a maximum at 48 hours.

In the case of L. reuteri , the growth of lactic acid bacteria hardly appeared until 18 hours at 25℃, and then it started to grow, so it can be confirmed that the cell growth of lactic acid bacteria is slow at low temperature. In addition, it can be seen that at 30° C., the highest value of lactic acid bacteria growth is high, but the production of lactate is low, and at 37° C., although the maximum value of lactic acid bacteria growth is low, the production of lactate and ethanol is high. From this, it can be seen that L. reuteri is a strain whose fermentation product and growth rate change significantly according to temperature, and is a strain that performs heterolactic fermentation to produce acetate and ethanol together with lactate.

<Experimental Example 2> Heat resistance analysis

5mL each of MRS medium was dispensed into test tubes, and 1% of the strain stored in a -80°C ultra-low temperature freezer was inoculated and cultured for 12 hours at 80rpm in a 30°C shaking incubator. 1 mL out of 5 mL of the culture solution was transferred to a test tube in which 4 mL of MRS was dispensed, and 5 mL was incubated at 30° C. in a shaking incubator at 80 rpm for 12 hours to measure OD.

Dispense 3mL of the remaining 4mL of the culture solution into 1.5mL tubes at a time of 1mL, and place these three tubes in a heat block (VWR Scientific, PA, USA) and water bath (DAIHAN Scientific) at 45℃, 55℃, and 65℃, respectively, for 3 hours. Co., Seoul, Korea). After heat treatment, the test tube was re-inoculated in MRS medium dispensed by 4 mL each, incubated for 12 hours at 80 rpm in a shaking incubator at 30 ° C.

Referring to FIG. 44 , L. fermentrum had an OD value of 5.94 when no heat shock was applied. When heat shock was given at 45° C., it was 5.74, 55° C., and when heat shock was given, it was 2.18, 65° C. When heat shock was given, it was measured as 1.42. Therefore, 96.63% survived at 45°C, 36.7% at 55°C, and only about 24% at 65°C.

In L. plantarum , 10.88 was measured when no heat shock was applied, 4.5 when 45 ℃ heat shock was given, 3.16 when 55 ℃ heat shock was given, and 2.15 when 65 ℃ heat shock was applied. Therefore, 41.4% survived at 45°C, 29.0% at 55°C, and only 19.8% at 65°C.

In L. rhamnosus , 11.8 was measured when no heat shock was applied, 8.86 when heat shock was applied at 45°C, 4.97 when heat shock was applied at 55°C, and 2.63 when heat shock was applied at 65°C. Therefore, 75.1% survived at 45°C, 42.1% at 55°C, and only 22.3% at 65°C.

L. mesenteroides measured 3.54 when no heat shock was applied, 3.02 when the heat shock was applied at 45℃, 2.8 when the heat shock was applied at 55℃, and 1.16 when the heat shock was applied at 65℃. Therefore, at 45°C, 85.3% survived, at 55°C, 79.1%, and at 65°C, only about 32.8% survived.

In L. acidophilus , 10.96 was measured when no heat shock was applied, 5.85 when the heat shock was applied at 45℃, 2.31 when the heat shock was applied at 55℃, and 1.33 when the heat shock was applied at 65℃. Therefore, 53.4% survived at 45°C, 21.1% at 55°C, and only 12.1% at 65°C.

In L. reuteri , 7.43 was measured when no heat shock was applied, 6.23 when heat shock was applied at 45℃, 1.37 when heat shock was applied at 55℃, and 1.054 when heat shock was applied at 65℃. Therefore, 83.8% survived at 45°C, 18.4% at 55°C, and only 14.2% at 65°C.

Through this, the five strains of L. fermentrum , L. rhamnosus , L. mesenteroides , L. acidophilus , and L. reuteri are strains that survive more than half of them even after applying heat shock at 45°C, and L. plantarum is relatively weak to heat. It can be confirmed that it is a strain. In addition, most strains showed a significant decrease in survival rate after applying heat shock at 55°C and 65°C, but L. mesenteroides showed high survival rate even when heat shock at 55°C was applied, confirming that heat resistance is higher than that of other strains. can

<Example 1> Preparation of fermented beverage

Carrots and bok choy were inoculated with various lactic acid bacteria to prepare fermented beverages, and then the characteristics of the fermented beverages were analyzed. First, after reviewing the appropriate mixing ratio of dried carrots and dried bok choy to be used in the manufacture of carrots and bok choy fermented beverages, 6 kinds of lactic acid bacteria were inoculated into the carrot and bok choy fermented stock solutions, and colonies were formed for physicochemical analysis of the fermented beverages thus prepared. Unit (colony forming unit, CFU), pH, HPLC was measured. The low-temperature storage properties of lactic acid bacteria in the fermented beverage were evaluated by measuring the number of viable cells at regular intervals while storing the beverage fermented for 72 hours at 4°C.

1. Material

The main ingredients used in the production of fermented beverages were dried carrots and dried bok choy sold by Purun Nongsan Co., Ltd., which are shredded into 2mm pieces that are easy to distribute and store. For the peach and plum juice concentrate used as a sugar source, Wellfine Co., Ltd.'s 'Jinhanwell Darker Peach Plum' product was used. For the strains used for physicochemical characterization, 6 strains ( Lactobacillus fermentum KACC11441, Lactobacillus plantarum KACC11451, Lactobacillus rhamnosus KACC11953, Leuconostoc mesenteroides KACC12312, Lactobacillus acidophilus KACC12419, Lactobacillus reuteri KACC11452) purchased from KACC were used. As a medium for lactic acid bacteria, MRS broth and Bacto agar (Difco, MI, USA) were used.

2. Review of proper mixing ratio of carrots and bok choy

An experiment was conducted to find an appropriate mixing ratio of the carrot and bok choy fermented undiluted solutions to be used in the manufacture of fermented beverages. The ratio of dried carrots to dried bok choy in 100mL of water was measured under the following 4 conditions: (10g dried carrots: 10g dried bok choy)(A), (10g dried carrots: 5g dried bok choy)(B), (10g dried carrots: dried bok choy) 1g)(C), (Dried carrot 5g: Dried bok choy 1g)(D) and ground in a blender for 1 minute, and then color and palatability were compared.

45, (Dried carrot 10g: dried bok choy 1g)(C), (dried carrot 5g: dried bok choy 1g)(D), (dried carrot 10g: dried bok choy 5g) (B), (dried carrot 10g: 10 g of dried bok choy) (A), a bright orange undiluted solution was produced, and a dilute undiluted solution was generated in the order of (D), (C), (B), and (A). Accordingly, it was judged that the mixing ratio of (10 g dried carrots: 1 g dried bok choy) (C) was appropriate for the production of a rich fermented beverage with bright color and palatability. produced.

3. Fermented beverage manufacturing

The fermented undiluted solution for making fermented beverages was prepared by adding 10 g of dried carrots and 1 g of dried bok choy to 100 mL of purified water, pulverizing them for 1 minute using a mixer, and sterilizing them at 121°C for 20 minutes under high pressure. Dangwon was injected by mixing 15 g of plum, peach juice concentrate, and 20 mL of purified water. Six strains to be used for beverage fermentation were inoculated with a 1% concentration of glycerol strain stock in 5mL of MRS medium contained in a test tube, and cultured overnight at 80rpm in a shaking incubator (Vision Scientific, Bucheon, Korea). After the growth of the bacteria, the culture medium was centrifuged at 13,500 rpm for 5 minutes to remove the supernatant, and only the cells were taken and used. The absorbance of each strain was measured under 600 nm conditions. The absorbance of 1 mL of the culture solution was adjusted to 0.1 and inoculated into the fermentation stock solution. 0.85% NaCl solution was used for cell dilution. All fermented beverages were fermented at 30° C. and 80 rpm for 72 hours ( FIG. 46 ).

<Experimental Example 3> Analysis of physicochemical properties of fermented beverages

1. Analysis method

1) Measurement of viable cell count

In order to measure the growth of lactic acid bacteria in the fermented beverage prepared according to Example 1, 1 mL of the fermented solution was diluted through decimal dilution and then spread on an MRS agar plate medium. Plates were cultured at 30°C for 48 hours, then the number of grown colonies was measured and multiplied by a dilution factor to calculate the number of viable cells in the culture solution as CFU.

2) pH

In order to observe the change in pH in the fermented beverage during fermentation of each strain, 1 mL of the fermentation broth was taken every predetermined time using a Beckman 390 pH meter (Beckman Coulter Inc., CA, USA) to measure the pH.

3) HPLC

For quantitative analysis of the fermentation product of each strain, a sample of 1 mL of the fermentation broth was taken, centrifuged at 13,500 rpm for 10 minutes, and then 700 μL of the supernatant was filtered with a 2 μm syringe filter and analyzed by HPLC. The HPLC instrument used for the analysis was an Agilent 1260 Infinity Ⅱ (Agilent, Santa Clara CA, USA), and the column was an Aminex HPX-87H Column (300 mm×7.5 mm, 9 μm, Bio-Rad, CA, USA). As the mobile phase, a solvent of 0.005 M sulfuric acid was used, and the solvent condition was analyzed by flowing a solvent of 0.005 M sulfuric acid for 30 minutes. The temperature of the column oven was maintained at 60° C., the sample injection amount was 10 μL, and the flow rate was 0.6 mL/min. All samples were quantitatively analyzed using RID (refractive index detector).

2. Analysis Results

1) Uninoculated control (CONTROL) stock solution

Referring to Figures 47 and 48, in the case of the fermentation stock CONTROL that was not inoculated with the strain, there was no change in any of the values. Through this, it can be confirmed that the high-pressure sterilization of the carrot and bok choy fermented undiluted solution was well performed. In addition, the fermentation stock solution contained about 60 g/L of glucose, about 50 g/L of sucrose, and about 16 g/L of fructose, and it was confirmed that it came from the plum and peach juice concentrates used as sugar sources.

Therefore, it was judged to be suitable for use in strain fermentation experiments as a food hygienically appropriate fermentation stock solution. In addition, despite autoclaving at 120° C., it can be seen that the color does not change much and has a thin tomato color.

2) Lactobacillus Fermentum ( Lactobacillus fermentum ) inoculated fermented beverage

Referring to FIG. 49, L. fermentum did not grow almost during the initial 24 hours, but it was found to grow continuously until 72 hours thereafter. The pH value of L. fermentum decreased continuously for 72 hours, and the decrease was highest in the initial 24 hours.

Referring to FIG. 50 , L. fermentum showed a large decrease in glucose and sucrose compared to fructose during the initial 24 hours, and a clear decrease in fructose until 72 hours thereafter. Ethanol was almost unchanged for 72 hours, and acetate and lactate increased continuously for 72 hours, finally recording 1.4 g/L and 2.8 g/L.

3) Lactobacillus plantarum ( Lactobacillus plantarum ) inoculated fermented beverage

Referring to FIG. 51 , L. plantarum was shown to grow rapidly during the initial 24 hours, and then grow steadily until 72 hours. The pH value of L. plantarum decreased rapidly during the initial 24 hours and then decreased steadily until 72 hours thereafter.

Referring to FIG. 52, L. plantarum showed a continuous decrease in saccharides for 72 hours. During 72 hours, sucrose, fructose, and glucose all tended to decrease, and during the first 24 hours, glucose decreased the most. Ethanol and acetate hardly increased during 72 hours, and lactate increased continuously for 72 hours, finally recording 6.3 g/L.

4) Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) inoculated fermented beverage

Referring to Figure 53, L. rhamnosus was shown to grow rapidly for the initial 24 hours and then slowly grow up to 72 hours. The pH value of L. rhamnosus showed a rapid decrease up to 48 hours and then slowly decreased after 72 hours.

Referring to FIG. 54, L. rhamnosus continuously decreased saccharides for 72 hours, and sucrose, fructose, and glucose all decreased. During the first 24 hours, glucose and sucrose were significantly decreased compared to fructose, and after 48 hours, fructose decreased the most. Ethanol hardly increased during 72 hours, and acetate and lactate increased continuously for 72 hours, finally recording 2.2 g/L and 3.7 g/L.

5) Leukonostok mecenteroides ( Leuconostoc mesenteroides ) inoculated fermented beverage

Referring to FIG. 55, L. mesenteroides continued to grow for 72 hours, and in particular, it was found to grow rapidly after 24 hours. The pH value of L. mesenteroides decreased rapidly after the initial 24 hours and continued to decrease until 72 hours.

Referring to FIG. 56 , L. mesenteroides showed a continuous decrease in saccharides for 72 hours, and all of sucrose, fructose, and glucose tended to decrease. In the first 24 hours, sucrose was decreased the most, and the decrease in glucose and fructose was high until 48 hours thereafter. Acetate and ethanol were almost unchanged for 72 hours, and lactate continued to rise, recording 3.7 g/L at 72 hours.

6) Lactobacillus acidophilus ( Lactobacillus acidophilus ) inoculated fermented beverage

Referring to FIG. 57 , L. acidophilus was continuously grown for 72 hours, and the pH value was continuously decreased for 72 hours, and in particular, it was shown to decrease rapidly after the initial 24 hours.

Referring to FIG. 58, L. acidophilus showed a continuous decrease in saccharides for 72 hours, and all of sucrose, fructose, and glucose tended to decrease, and glucose showed the greatest decrease until the initial 24 hours. Acetate and ethanol were almost unchanged for 72 hours, and lactate continued to rise, recording 4.1 g/L at 72 hours.

7) Lactobacillus reuteri ( Lactobacillus reuteri ) inoculated fermented beverage

Referring to FIG. 59 , L. reuteri grew up to 48 hours, but then stopped growing and decreased after 72 hours. The pH value of L. reuteri decreased at 72 hours compared to 0 hours, but the decrease was very small at 0.01.

Referring to FIG. 60 , L. reuteri showed a continuous decrease in saccharides for 72 hours, and all of sucrose, fructose, and glucose were decreased, and the amount of glucose decreased the most until the initial 72 hours. During 72 hours, there was little change in acetate and ethanol, and little change in lactate.

Combining the above results, it can be confirmed that the pH of all strains decreases as the number of live cells increases during fermentation of the fermented beverage. This is expected to depend on the characteristics of lactic acid bacteria that produce lactate as a metabolite. As the bacteria grow, the production of lactate also increases and the pH decreases accordingly.

L. fermentum showed little increase in ethanol and an increase in acetate and lactate during fermentation of carrot and bok choy fermented stock solution, but in characterization using MRS medium, it can be confirmed that it is a hetero type that produces an excess of ethanol. . An increase in the number of cells and a decrease in pH occur rapidly in the initial stage of fermentation, but in the case of a heterotype, it may be determined that it is not suitable for manufacturing a non-alcoholic fermented beverage because it produces ethanol.

It can be confirmed that L. plantarum produces only lactate and is a Homo type. Since the production rate of the initial cells and lactate is very fast, there is an advantage in the production of fermented beverages. Therefore, L. plantarum can be determined as a suitable strain for fermented beverages.

It can be confirmed that L. rhamnosus is also a Homo type lactic acid bacterium. Since the initial production of cells and the production of lactate and acetate are large, rapid production is possible when manufacturing fermented beverages and the reproduction of harmful bacteria can be effectively controlled, so it can be judged as a suitable strain for manufacturing fermented beverages.

L. mesenteroides did not grow well in the early stage, but showed an increase after 24 hours, showing that it is not suitable for the production of fermented beverages, where the production of a large number of cells and lactate in the initial stage is recommended. In addition, according to Figure 26, since it can be seen that it is a heterotype lactic acid bacteria, there is a possibility of ethanol production, so it can be determined that it is not suitable for manufacturing a non-alcoholic fermented beverage.

L. acidophilus shows little initial production of cells and little decrease in pH. This may be judged to be unsuitable as a strain for fermented beverages because productivity decreases during production of fermented beverages and may form fermented beverages susceptible to harmful bacteria.

L. reuteri showed little decrease in pH and little growth of cells within 72 hours of fermentation, and almost no increase in lactate, which is thought to be because the fermented beverage manufacturing stock solution is not suitable for the growth of L. reuteri. do. Accordingly, it may be determined that the carrot and bok choy fermented beverages are not suitable for manufacturing.

The strain with the fastest growth rate during the initial 24 hours and the most grown strain in the final 72 hours was L. plantarum, and the strain with the least growth for 72 hours was L. reuteri . After 72 hours, the strain with the lowest pH was L. plantarum, and the strain with the highest pH was L. reuteri . L. plantarum is considered appropriate if you want to secure a large number of probiotics in a short time or maintain a low pH to prevent the growth of harmful bacteria.

Judging from this, it can be judged that L. plantarum strain is appropriate for securing probiotics and suppressing the growth of harmful bacteria in fermented drinks of carrots and bok choy. The strains that produced the most lactate for 72 hours were L. plantarum , L. rhamnosus , and L. mesenteroides in the order, and L. plantarum had the highest rate of lactate production compared to sugar source consumption for 72 hours (47%).

Therefore, it was determined that strains such as L. plantarum KACC11451 and L. rhamnosus KACC11953 were suitable for the production of lactic acid bacteria fermented beverages that ultimately require the production of lactate to secure probiotics and suppress the growth of harmful bacteria.

<Experimental Example 4> Evaluation of low-temperature storage properties of fermented beverages

1. Evaluation method

Each fermented beverage was fermented for 72 hours and stored at 4° C., and low-temperature storage properties were evaluated through live cell count analysis. CFU measurement was used for live cell count analysis. For CFU measurement, 1 mL of the fermented beverage in storage was diluted using the decimal dilution method, and then spread on an MRS agar plate. After the plate was incubated at 30°C for 48 hours, the number of grown colonies was counted to measure the number of viable cells. The final CFU was calculated by multiplying the number of colonies by the dilution factor.

2. Evaluation results

61 to 66, L. fermentum , L. rhamnosus , L. acidophilus maintained a CFU value higher than the initial CFU value from the start of low-temperature storage to 168 hours, and the pH was maintained below 3.8. According to this result, it can be expected that the number of live probiotics can be maintained for more than 7 days during distribution after production of the actual fermented beverage, and it can be determined that the growth of harmful bacteria can be suppressed due to the low pH. Accordingly, it can be confirmed that the strain is suitable as a strain for production of fermented beverages that must maintain the number of live probiotics even when stored at a low temperature.

L. plantarum , L. mesenteroides , and L. reuteri showed a decrease in CFU after 168 hours from the start of cold storage. It can be expected that the number of live probiotics at the time of shipment cannot be maintained after 7 days in the distribution process after production of the actual fermented beverage, and it can be confirmed that L. reuteri is difficult to effectively prevent the growth of harmful bacteria as the pH is maintained above 3.9. there was. Therefore , it can be judged that L. plantarum , L. mesenteroides , and L. reuteri are relatively unsuitable for use in the production of fermented beverages that need to maintain the number of live prebiotics for a long time.

<Example 2> Lactobacillus rhamnosus for optimizing fermentation conditions ( Lactobacillus rhamnosus ) Production of fermented beverages using strains

After characterization of the strain, the final lactic acid bacteria strain for commercialization of carrot and bok choy fermented drinks was selected through characterization of carrot and bok choy fermented beverages and evaluation of low-temperature storage properties. In the characterization analysis of carrots, bok choy, and fermented beverages using 6 strains, L. plantarum , L. rhamnosus, etc. were judged to be suitable strains for the production of fermented beverages, and L. fermentum , L. rhamnosus , L. acidophilus for low-temperature storage properties. was determined to be a suitable strain for the production of fermented beverages. The strain determined to be suitable for the production of fermented beverages in both the characterization and low-temperature storage evaluation was Lactobacillus rhamnosus , and an experiment was conducted for optimizing the fermentation conditions of the fermented bok choy drink using this. Beverage fermentation was performed by varying the concentration of the sugar source, the culture temperature, and the initial strain concentration. The effect on fermentation was analyzed by measuring the number of viable cells, pH, and HPLC of the fermentation broth. Sensory evaluation was performed on beverages fermented under different conditions. L. rhamnosus was added to carrots and bok choy to prepare granulated products with long shelf life and easy storage. Sensory evaluation and storage performance evaluation of the prepared granule product were performed.

1. Material

Carrots and bok choy, which are the main ingredients used in making fermented beverages, were made from dried carrots and dried bok choy from Purun Nongsan. For the peach and plum juice concentrate used as a sugar source, Wellfine Co., Ltd.'s 'Jinhan Well Darker Peach Plum' was used. As the strain used for manufacturing fermented beverages and manufacturing granulated products, Lactobacillus rhamnosus acquired from KACC was used. Carrot powder, bok choy powder, L. rhamnosus fermentation solution, lactose, dextrin, glucose, corn starch, refined salt, DHA, powdered sugar, nucleic acid IG and purified water were used to manufacture carrot and bok choy granules containing lactic acid bacteria. MRS broth and bacto agar (Difco, MI, USA) were used as a medium for lactic acid bacteria growth.

2. Fermented beverage manufacturing

The fermented undiluted solution for making fermented beverages was prepared by adding 10 g of dried carrots and 1 g of dried bok choy to 100 mL of purified water, pulverizing them for 1 minute using a mixer, and sterilizing them by heating at 121° C. for 20 minutes. The strain to be used for beverage fermentation was inoculated with a 1% concentration of the selected strain stored at -80 ° C in 5 mL of MRS medium contained in a test tube, and cultured overnight at 80 rpm in a shaking incubator (VS-8480, Vision Scientific, Bucheon, Korea). After the growth of the bacteria, the culture medium was centrifuged at 13,500 rpm for 5 minutes (Smart 12, Hanil Science Inc., Kimpo, Korea) to remove the supernatant, and then only the cells were taken and inoculated with the entire amount.

To check the fermentation characteristics according to the concentration of the sugar source , add 20 mL of purified water, 15 g of sugar source, 10 mL of purified water, 25 g of sugar source, and 35 g of sugar source to three fermentation stock solutions inoculated with L. rhamnosus, respectively. 30℃, 80rpm was fermented for 72 hours.

To check the fermentation characteristics according to the culture temperature, a solution of 20 mL of purified water and 15 g of sugar source was added to three fermentation stock solutions inoculated with L. rhamnosus , and fermented for 72 hours at 25 ° C, 30 ° C, and 37 ° C at 80 rpm, respectively.

In order to check the fermentation characteristics according to the initial strain concentration, a solution of 20 mL of purified water and 15 g of sugar source was added to three fermentation stock solutions, and L. rhamnosus collected by centrifugation after overnight fermentation in 5 mL, 25 mL, and 50 mL MRS broth, respectively, was inoculated. It was fermented for 72 hours at 30° C. and 80 rpm ( FIG. 67 ).

<Experimental Example 5> Analysis of optimal fermentation conditions for fermented beverages

1. Analysis method

1) Measurement of viable cell count

In order to measure the growth of lactic acid bacteria in the fermented beverage prepared according to Example 2, 1 mL of the fermented broth was diluted through decimal dilution and spread on an MRS agar plate. The plate was incubated at 30°C for 48 hours, and the number of grown colonies was counted to measure the number of viable cells. The final CFU was obtained by multiplying the dilution factor.

2) pH

In order to observe the change in pH in the fermented beverage during fermentation under each condition, a 1 mL sample of the fermentation broth was taken and pH was measured using a Beckman 390 pH meter (Beckman Coulter Inc., CA, USA).

3) HPLC

For quantitative analysis of fermentation products under each condition, a sample of 1 mL of fermentation broth was taken, centrifuged at 13,500 rpm for 10 minutes (Smart 12, Hanil Science Inc., Kimpo, Korea), and 700 μL of the supernatant was filtered with a 2 μm syringe filter. It was analyzed by high performance liquid chromatograph (HPLC). The HPLC instrument used for the analysis was an Agilent 1260 Infinity Ⅱ (Agilent, Santa Clara CA, USA), and the column was an Aminex HPX-87H Column (300 mm×7.5 mm, 9 μm, Bio-Rad, CA, USA). As the mobile phase, a solvent of 0.005 M sulfuric acid was used. The temperature of the column oven (Agilent G7130A) was measured at 60°C, the sample injection amount was 10 μL, and the flow rate was 0.6 mL/min. The standard solution was quantitatively analyzed using RID (refractive index detector).

2. Analysis Results

1) Confirmation of effect of sugar source concentration on fermentation of fermented beverages

68 to 70, the highest growth of bacteria during 72 hours of fermentation was a fermented beverage to which 10 mL of purified water and 25 g of sugar were added, and 9.24 log CFU/mL was recorded. In the fermented beverage to which a mixture of 20 mL of purified water and 15 g of sugar source was added, 9.18 log CFU/mL was recorded during 72 hours of fermentation, and 9.03 log CFU/mL was recorded in the fermented beverage to which 35 g of sugar was added. The highest bacterial growth up to 24 hours after fermentation was a fermented beverage containing a mixture of 10 mL of purified water and 25 g of sugar source.

It is important to secure a lot of probiotics for fermented drinks with carrots and bok choy, and for the efficiency of mass production, the growth rate of bacteria in the early stage of fermentation is important. Therefore, it was judged that it is appropriate to ferment carrot and bok choy fermented drinks by adding a solution of 10 mL of purified water and 25 g of sugar source to the fermented stock solution.

2) Confirmation of influence of culture temperature on fermentation of fermented beverages

71 to 73, the growth rate of the bacteria was the slowest at 25°C, and the growth rate of the bacteria during the initial 24 hours was the highest at 37°C, and the amount of increase in lactate and the decrease in pH were also the highest during the same time. . In the production of probiotic fermented beverages, increasing the number of cells within a short time can increase productivity. Accordingly, it was determined that culturing at 37° C. was most suitable for the manufacture of fermented drinks with carrots and bok choy.

3) Confirmation of effect of initial strain concentration on fermentation of fermented beverages

74 to 76 , it can be seen that the increase in the amount of cells, the decrease in pH, and the lactate production are the highest in the fermented beverage inoculated with 50 mL of the cells. In the fermented beverage inoculated with 5 mL of cells, the increase in the amount of cells, the decrease in pH, and the lactate production were the lowest. According to this, in order to grow many probiotic cells and to suppress the growth of harmful bacteria by rapidly decreasing the pH, it is appropriate to inoculate 50 mL of the fermented drinks with carrots and bok choy.

However, in order to increase the inoculum of lactic acid bacteria, it is necessary to pre-culture a larger amount of cells, which also consumes cost, which may be uneconomical in the production of fermented beverages. Compared to 50mL of cells, 25mL of inoculated fermented beverage produces lactate more efficiently than 50mL of inoculated fermented beverage. It is considered to be more suitable for the production of fermented beverages.

<Experimental Example 6> Sensory evaluation of fermented beverages

The sensory evaluation of carrot and bok choy fermented beverages was performed using a 5-point scoring method for evaluation items such as sweetness, sourness, flavor, richness, and personal preference for 10 sensory personnel with experience in sensory evaluation.

Table 1 shows the sensory evaluation results of the carrot and bok choy fermented beverages prepared according to Example 2 of the present invention.

Referring to Table 1 above, when comparing according to the concentration of the sugar source, the fermented beverage to which 35 g of the sugar source was added received the highest score in sweetness, flavor, concentration, and personal preference. It is also the highest score in all comparison groups.

When comparing by fermentation temperature, the fermented beverage fermented at 37°C received the highest score in acidity and personal preference, and the fermented beverage fermented at 25°C received the highest score in aroma and concentration.

When compared according to the initial strain concentration, the fermented beverage inoculated with 50 mL of the bacteria received the highest score in sour taste and the lowest personal preference and flavor score overall.

According to these results, it seems that 35g of plum and peach juice concentrate is appropriate for the final fermented drink of carrots and bok choy. However, due to the nature of probiotic fermented beverages, it is predicted that marketing of the image of a healthy fermented beverage that is not sweet is necessary. Therefore, it is thought that the 25g sugar source concentration, which had the highest consumer preference after the 35g sugar source concentration with the highest sweetness, would be suitable as the final sugar source concentration.

The fermentation temperature seems to be appropriate at 37℃, which has the highest personal preference among the three conditions and can increase the sour taste of beverages. In the case of the initial cell concentration, it can be seen that the individual preference decreases as it increases from 5mL to 50mL, which is thought to be due to the excessive sour taste. As a result, based on the moderate increase in acidity and the rapid growth rate of the cells shown in FIG. 75, it is thought that 25mL cell inoculation is most appropriate for the manufacture of fermented drinks with carrots and bok choy.

Finally, according to the optimization of the fermentation conditions of the fermented beverage and the sensory evaluation test, the probiotic cell production capacity is excellent, it can effectively suppress harmful microorganisms, and when considering the marketing plan that meets the consumer's preference and the purpose of the lactic acid bacteria fermented beverage, L. For the production of fermented carrot and bok choy drinks using rhamnosus, a solution of 10 ml of purified water and 25 g of sugar source is added to the fermentation stock, inoculated with the cells produced in 25 ml of MRS broth, and fermentation at 37° C. is considered the most suitable.

<Example 3> Preparation and evaluation of carrot and bok choy granulated products containing lactic acid bacteria

1. Manufacturing method

The L. rhamnosus fermented broth used for the production of carrot and bok choy granulated products containing lactic acid bacteria was inoculated with the selected strain stored at -80 ° C in 400 mL of MRS medium in an Erlenmeyer flask at 1% concentration in a shaking incubator (VS-8480, Vision Scientific , Bucheon, Korea) was cultured overnight at 80 rpm. After the growth of the bacteria, the culture medium was centrifuged at 13,500 rpm for 5 minutes (Smart 12, Hanil Science Inc., Kimpo, Korea) to remove the supernatant, and only the cells were collected in 500 mL purified water. Carrot and bok choy granulated products were mixed in a high-speed mixer after measuring the raw materials according to the quantity. The mixed carrot stock solution was granulated using an extrusion granulator. The granulated carrot undiluted solution was dried at 65°C until the moisture content reached 2.5-3.5%. The dried granules were screened through a 5mm*5mm vibrating sieve. The selected granular product was passed through a metal detector (Fe: 1.2 mm, SUS: 1.5 mm) and then packaged in small packages (FIG. 77).

2. Blending ratio

1) Carrot-flavored granulated product containing lactic acid bacteria fermented liquid

As a result of confirming the physical properties of the formulation (before granulation) containing 10% of the lactic acid bacteria fermentation broth as a preliminary experiment, it was determined that granules could not be formed because it was too sticky. Thus, an appropriate mixing ratio was confirmed. The valence ratio may vary depending on the type of raw material.

Table 2 is a table showing the ingredients and mixing ratio of the carrot-flavored granulated product according to Example 3 of the present invention.

Referring to Table 2, when the content of 'carrot powder', which is the raw material powder of agricultural products, is below a certain ratio, it appears that the granular form is maintained when it passes through the granulation network and even after drying. appropriateness can be checked. When a lot of 'carrot powder' was contained, not only did it pass through the granulation network with difficulty, but the passed granules were not maintained and loosened. This is an excipient in the extruded granulation method such as lactose, dextrin, and glucose, which acts as a carrier. It also means that the content of raw materials being used must be maintained over a certain level.

2) Cheonggyeongchae flavor granule product containing lactic acid bacteria fermented liquid

As with carrots, as a result of confirming the physical properties of the formulation (before granulation) containing 10% of the lactic acid bacteria fermentation broth in the prior experiment, it was judged that granules could not be formed because it was too sticky. % was fixed to confirm an appropriate mixing ratio. The valence ratio may vary depending on the type of raw material.

Table 3 is a table showing the ingredients and mixing ratio of the bok choy flavor granule product according to Example 3 of the present invention.

Referring to Table 3, when the content of 'bok choy powder', which is the raw powder of agricultural products, is below a certain ratio, it appears that the granule form is maintained when it passes through the granulation network and even after drying, so Sample #3 is the most appropriateness can be checked. When a lot of 'bok choy powder' was contained, not only did it pass through the granulation network with difficulty, but also the passed granules were not maintained and loosened. This is an excipient of the extruded granulation method such as lactose, dextrin, and glucose, which acts as a carrier. It also means that the content of raw materials being used must be maintained over a certain level.

3. Evaluation

The sensory evaluation of carrot and bok choy granulated products containing lactic acid bacteria was performed by describing the evaluation of the product after tasting the granulated product targeting 4 sensory personnel. The number of viable cells was measured to evaluate the shelf life of the granule product. To measure the number of viable cells in the granule product, 1 g of carrot and bok choy granulated products were mixed with 5 mL of MRS medium contained in a test tube, 1 mL of which was taken, diluted through decimal dilution, and smeared on an MRS agar plate to measure CFU. The final CFU was obtained by multiplying the dilution factor.

1) Sensory evaluation

Carrots and bok choy granulated products containing lactic acid bacteria have a somewhat rough texture and do not have good loosening properties in the mouth. It has a salty taste so that it can be added to seasoning seasonings. It has a mild fishy taste due to the addition of DHA, which is an auxiliary ingredient, and a umami taste due to nucleic acid IG. However, it was judged that when it was used in the form of furikake together with other ingredients as a material for secondary processing, it could be consumed in various forms harmoniously. Lactobacillus granule products containing carrots and bok choy, which are rich in various physiologically active ingredients and fiber, are synbiotics containing both probiotics and prebiotics. It is believed that it could be a possible food ingredient.

2) Storage evaluation

Referring to FIG. 78 , the number of viable cells gradually decreased when stored at room temperature from 2 days after production to 12 days. Therefore, it is expected that the number of live bacteria will decrease continuously when stored at room temperature for a long time. Since the shelf life of general granule products is 12 months, it is expected that the shelf life of granules containing lactic acid bacteria will be shorter than this. In order to prevent a decrease in the number of viable cells, it is considered desirable to avoid direct sunlight and store at room temperature in a cool place. The final moisture content of the granules is 2.5~3.5%, and reabsorption of moisture is expected during small packaging. In bulk packaging, it is necessary to enclose a desiccant to prevent moisture absorption.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (14)

preparing a dried fruit and vegetable stock solution;
adding water and a sugar source to the prepared stock solution; and
Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), Lactobacillus plantarum ( Lactobacillus plantarum ), or a mixed lactic acid bacteria thereof was inoculated into the medium and cultured, followed by fermentation by mixing the cultured lactic acid bacteria in the stock solution to which the water and the sugar source were added. A method of producing a lactic acid bacteria fermented food comprising a; The method of claim 1,
The step of preparing the dried fruit and vegetable stock solution,
A method for producing lactic acid bacteria fermented food, characterized in that the dried fruits and vegetables are put in water and pulverized, and then sterilized at 110 to 130° C. for 10 to 30 minutes. The method of claim 1,
The dried fruits and vegetables include dried carrots and dried bok choy,
The dried carrots and dried cheonggyeongchae (5 to 15): A method for producing lactic acid bacteria fermented food, characterized in that it is included in a weight ratio of 1. The method of claim 1,
The sugar source includes peach or plum juice concentrate,
The water and the sugar source are 1: (1 to 4) a method for producing a fermented lactic acid bacteria food characterized in that mixed in a weight ratio. The method of claim 1,
The fermenting step is
After inoculating and culturing the lactic acid bacteria in 20 to 50 mL of MRS medium, the method for producing a lactic acid fermented food, characterized in that the fermentation is performed at 30 to 40 ℃ by mixing the lactic acid bacteria obtained by centrifugation. A fermented lactic acid bacterium food prepared according to any one of claims 1 to 5. 7. The method of claim 6,
The lactic acid bacteria fermented food,
Lactobacillus fermented food, characterized in that it is a lactic acid bacteria fermented fruit and vegetable beverage. Preparing a fruit and vegetable stock solution by mixing the fruit and vegetable powder and Lactobacillus rhamnosus or Lactobacillus plantarum lactic acid bacteria, or a lactic acid bacteria fermentation broth containing at least one of the lactic acid bacteria;
granulating the prepared fruit and vegetable stock solution; and
Drying the granulated fruit and vegetable stock solution; Lactic acid bacteria-containing granular food manufacturing method comprising. 9. The method of claim 8,
The fruit and vegetable powder,
Based on 100 parts by weight of the whole food, 15 to 30 parts by weight may be included,
The lactic acid bacteria-containing granular food manufacturing method, characterized in that the fruit and vegetable powder and the lactic acid bacteria or fermented lactic acid bacteria (2 to 4) are mixed in a weight ratio of 1. 9. The method of claim 8,
The fruit and vegetable powder,
Carrots, bok choy, avocado, kale, celery, and a method for manufacturing a granular food containing lactic acid bacteria, characterized in that the powder of one or more fruits and vegetables selected from the group consisting of beets. 9. The method of claim 8,
The drying step is
Lactic acid bacteria-containing granular food manufacturing method, characterized in that it is carried out at 60 to 70 ℃ so that the water content of the granulated fruit and vegetable stock solution is 2 to 4 wt%. 9. The method of claim 8,
The fruit and vegetable undiluted solution,
Lactobacillus-containing granular food manufacturing method, characterized in that it further comprises one or more selected from the group consisting of lactose, dextrin, glucose, corn starch, refined salt, DHA, powdered sugar and nucleic acid IG. A granular food containing lactic acid bacteria prepared according to any one of claims 8 to 12. 14. The method of claim 13,
The food is
A granular food containing lactic acid bacteria, characterized in that it has a synbiotic effect.

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