KR101224598B1 - Printable paste for microbial time-temperature integrator and producing method for the same - Google Patents

Abstract

PURPOSE: Printed paste for a microorganism type TTI, a manufacturing method thereof, and the microorganism type TTI including the same are provided to mass produce lactobacillus, and to enhance visibility and dry ability. CONSTITUTION: Printed paste for a microorganism type TTI contains lactobacillus micro-stereotaxic particles which are fixed to 1.5 - 2.5%(w/v) of sodium alginate. Based on the amount of the paste, the amount of the lactobacillus microstereotaxic particles is 10%(w/v). The printed paste additionally includes a mixed indicator solution, glycerol, a nutrient component, pulluran, cryoprotectant, and low temperature cryostabilizer. Based on the printed paste, the content of flurane is 5-15%(w/v).

Description

Printable paste for Microbial Time-Temperature Integrator and Producing method for the same}

The present invention relates to a printing paste for microbial time-temperature integrator (TTI), a method for producing the same, and a microbial TTI including the printing paste.

More specifically, the present invention relates to a printing paste for microbial TTI, which contains micro-immobilized lactic acid bacteria immobilized on sodium-alginate.

Food safety is of greatest interest throughout the food industry. Most consumers are demanding a way to assess the safety of their food when they buy perishable food. To date, the most common method is to check the date of manufacture. Therefore, there is a need for the development of a method that can easily, quickly and simply measure the safety of food from production to final consumption.

Time-temperature integrators (TTIs) are a simple, inexpensive, easy-to-use tool that allows you to monitor, record, and accumulate the overall effect of a full or partial temperature history from food production to final consumption. an easy device (Taoukis, PS, & Labuza, TP (1989). Applicability of time-temperature indicators as shelf life monitors of food products. Journal of Food Science , 54 (4), 783-788; Giannakourou, MC, Koutsoumanis, K., Nychas, GJ, & Taoukis, PS (2005). Field evaluation of the application of time temperature integrators for monitoring fish quality in the chill chain. International Journal of Food Microbiology, 102 (3), 323-236). These are products that are labeled on the packaging of the product to keep an eye on the time-temperature history of perishable foods during distribution. The principle of operation of TTIs is mechanical, chemical, enzymatic, microbiological or photoreversible irreversible changes, which are expressed in the form of visible reactions such as modification of the TTI itself, color development or color shift. Most TTIs do not directly represent food quality changes. They only represent color changes when the food is exposed to inappropriate temperatures, where managers or consumers check the quality of the food. Microbial indicators, in which TTI's response is directly related to food deterioration, can represent food quality better than other TTIs. Microbial indicators have recently been commercialized, but are not fully commercialized due to cost and accuracy of reactions. To date, much research has been conducted on kinetics of TTI. However, research on methods and apparatus for the development of new microbial TTIs is insufficient.

When designing and manufacturing microbial TTIs, the first consideration is to ensure that the indicators show a clear, continuous and irreversible color change with temperature changes. In addition, the indicator should remain inactive normally and be activated when attached to food packaging. Usually deactivation of the indicator can be achieved through freezing, microimmobilization and compartmentalization techniques. Microimmobilization is a technique for capturing a solid, liquid or gas into a very small sealed capsule, in which the captured material releases the component at a controlled rate under special conditions (Shahidi, F., & Han, XQ (1993). Encapsulation of food ingredients. Critical Reviews in Food Science and Nutrition , 33 (6), 501-547). There are several examples of microimmobilization of lactic acid bacteria with polysaccharides to protect them from high concentrations of oxygen, acidic environments, freezing and refrigeration.

Lactic acid is a very important metabolite produced during the fermentation metabolism of lactic acid bacteria, the degree of acidification by lactic acid production is visualized by using an indicator, and a substance causing various color changes by the redox reaction may be applied thereto.

Modern printing technology requires high quality printing pastes. The most important part of the printing technology is highly dependent on the physical properties such as the viscosity, surface tension, non-foamability, good adhesion, fast drying properties, high image quality and irreversibility of the printing pastes. Generally, oily pastes are used for printing non-porous materials, but aqueous pastes are used for printing porous materials. The reaction layer of the microbial TTI should be stable, highly sensitive and selective. Appropriate adhesion of the texturing agent uniformly applied to the polymer film is essential to ensure the correct response of the time-temperature history indicator.

However, there are no studies on efficient printing pastes considering wettability and drying characteristics in microbial TTI.

Disclosure of Invention An object of the present invention is to provide a microbial TTI printing paste including the same, and a paste for microbial TTI in consideration of wettability and drying characteristics that influence print quality.

Still another object of the present invention is to provide a method of manufacturing a printing paste for microbial TTI suitable for the microbial TTI as described above.

In order to achieve the above object, the present invention provides a printing paste for microbial TTI, characterized in that it contains micro-immobilized lactic acid bacteria immobilized on sodium-alginate.

The present invention also provides a microbial TTI comprising the above-described printing paste for microbial TTI.

In addition, the present invention comprises the steps of 1) preparing CaCO ₃ -alginate beads 2) inoculating lactic acid bacteria in a medium containing the beads of 1) to cultivate a high concentration of lactic acid bacteria 3) 2) micro-fixing the lactic acid bacteria of 2); And 4) provides a printing paste manufacturing method for microbial TTI comprising the step of preparing a printing paste containing the fine immobilized particles of 3).

According to the present invention, a large amount of lactic acid bacteria can be cultured by using CaCO ₃ -alginate, the most suitable immobilized lactic acid microparticles can be prepared by adjusting the sodium-alginate content, and the most suitable printing paste can be provided by controlling the content of pullulan. In addition, it provides a microbial TTI with improved visibility and drying ability can be usefully used in the food industry.

; 5% 플루란-2.0% LCAMs,

; 10% 플루란 -2.0% LCAMs,

; 15% 플루란 -2.0% LCAMs)
도 3은 수용성 인쇄 페이스트의 수분 흡습 곡선을 도시한다. (■; 5% 플루란-2.0% LCAMs system, ◆; 10% 플루란 -2.0% LCAMs system, ▲; 15% 플루란 -2.0% LCAMs)
도 4는 미생물형 TTI의 색 변화를 나타내며 작은 사각형은 L a b 유니트의 색 변화를 나타낸다. ((A) free cell의 수용성 인쇄 페이스트의 색 변화, (B) 48시간 후 활성화된 인쇄 페이스트의 색 변화, (C) 72시간 후 활성화된 인쇄 페이스트의 색 변화, (D) 2.0% LCAMs 을 가진 수용성 인쇄 페이스트의 색, (E) 72시간 후 2.0% LCAMs 을 가진 활성화된 수용성 인쇄 페이스트의 색 변화) 1 shows a micrograph of LAB-loaded Ca-alginate microparticles (LCAM) by spray-fixed method. ((A) is the optical micrograph (OM), scanning electron micrograph (SEM) of LCAMs prepared using 1.5% (w / v) sodium-alginate, and (B) is 2.0% (w) (v) OM, SEM of LCAMs prepared using sodium-alginate, (C) is OM and SEM of LCAMs prepared using 2.5% (w / v) sodium-alginate)
FIG. 2 shows the weight loss and weight loss rates when the pullulan-LCAMs system was dried while heating at 10 ° C./min. ((A) weight loss curve, (B) speed of weight loss,

; 5% pullulan-2.0% LCAMs,

; 10% pullulan -2.0% LCAMs,

; 15% pullulan -2.0% LCAMs)
3 shows the moisture absorption curve of the water soluble printing paste. (■; 5% Fulan-2.0% LCAMs system, ◆; 10% Fulan -2.0% LCAMs system, ▲; 15% Fulan -2.0% LCAMs)
4 shows the color change of the microbial TTI and the small square shows the color change of the L ab unit. (A) color change of water-soluble printing paste in free cell, (B) color change of activated printing paste after 48 hours, (C) color change of activated printing paste after 72 hours, (D) 2.0% LCAMs Color of water soluble printing paste, (E) color change of activated water soluble printing paste with 2.0% LCAMs after 72 hours)

The present invention provides a printing paste for microbial TTI and microbial TTI using the same, which comprises fine immobilized lactic acid bacteria immobilized on sodium-alginate.

A time temperature indicator is a device that has one or more observable variability characteristics that progress at a rate proportional to temperature and time, indicating the overall or partial time-temperature history of the immediate surroundings that it is thermally contacting. The time temperature indicator (TTI) is a simple and inexpensive device usually designed as a label. When attached to perishable products, a properly designed and calibrated TTI monitors its time-temperature history and provides a straightforward visual overview of the product's exposure history over time-temperature to provide freshness to the product. Instruct. Thus, TTI is one of the most promising shelf life indication techniques.

The concept of TTI was developed to ensure the safety and quality of perishable products such as food and pharmaceuticals over their entire lifetime, from their manufacture or packaging to consumption by end users. The safety and quality of perishable products such as foods, drugs, vaccines and blood are largely dependent on the proper storage conditions during distribution and storage. Various factors such as gas composition, relative humidity and temperature affect their actual shelf life. The fact that the change in conditions affects the shelf life of this kind of product is not reflected in the label of the "best quality" type, which assumes suitable storage conditions. Based on various physical, chemical, enzymatic or microbial processes, the most frequently observed factor for alterations that occurred before the expiration date among all storage factors is temperature change. Thus, TTI technology can provide a simple means for managing the supply route of food and drugs. Since the color and / or other physical properties of the TTI change at a temperature dependent rate as a function of time, the color (or contrast) or any other variable visible property of the TTI label may be compared to a given comparison class to determine the " Gives an actual rating of "freshness". Since the TTI indicator can be designed to provide a clear answer such as "yes" or "no" with respect to the time temperature factor, it can provide an important "clear" answer and omit additional complex data checks. This is ideal for the Hazard Analysis Critical Control Points (HACCP), which focuses on real-time decision making and action.

Lactic acid bacteria used in the microimmobilization of the present invention can be used without limitation the lactic acid bacteria used in the art, in the present invention used Lactobacillus amylovorus KCCM 40431 as a preferred example.

“Alginate” of the present invention is an elastic impression material and has an irreversible property that changes from sol to gel but does not change from gel to sol due to chemical change, and refers to a material having hydrophilicity as a hydrocolloid impression material based on water. . The microimmobilization carrier of the present invention includes a porous polymer (agar, alginate k-carrageenan, polyacrylamide, chitosan, gelatin, collagen), porous metal membrane, polyurethane, silica gel, polystyrene, and cellulose triacetate. Without limitation, etc. can be used, and in the present invention, alginate is used as a preferred example.

By controlling the content of alginate it is possible to control the size of the microparticles and to control the number of particles of lactic acid bacteria to be fixed. Therefore, those skilled in the art to which the present invention belongs can adjust the content of alginate to produce the desired TTI, and preferably the alginate of the present invention may be included in 1.5 to 2.5% (w / v), more preferably 2.0%. (w / v) may be included.

The printing paste for microbial TTI of the present invention further includes a printing paste manufacturing material used in the art, preferably, a mixed indicator solution, a nutrient, pulluran, cryoprotectant, and a low temperature stabilizer ( cryostabilizer) and the like.

As the nutritional component of the present invention, MRS medium, MIC medium and Nutrient liquid medium can be used without limitation. In the present invention, MRS medium was used as a preferred example. The cryoprotectants or cryostabilizers include glycerol, skim milk, dextran, DMSO, inositol, sorbitol, mannitol, sucrose, corn syrup and trehalose. It can be applied without limitation, and in the present invention, glycerol was used as a preferred example.

Mixed indicators can be used without limitation, generally available indicators, such as Bromocresol green, Bromocresol purple, Chlorophenol red, Bromophenol blue, Congo red, as a preferred embodiment in the present invention 95% v / v ethyl alcohol and bromocresol purple ( 0.04%, w / v) mixture, 95% v / v ethyl alcohol and bromocresol green (0.05%, w / v) mixture, 95% v / v ethyl alcohol and ethyl orange (0.1%, w / v) Mixing indicators with a mixture of 1: 1: 1 were used.

The "pullulan" of the present invention is a food additive for increasing the adhesiveness and viscosity of foods, enhancing the emulsion stability, and improving the physical properties and feel of foods. It is widely used as a substitute for gum arabic and has excellent film forming ability. The pullulan may be preferably contained in an amount of 5 to 15% (w / v) based on the printing paste.

In another aspect, the present invention provides a microbial TTI comprising the printing paste for the microbial TTI.

As another embodiment of the present invention 1) step of preparing CaCO ₃ -alginate beads 2) inoculating lactic acid bacteria in the beads of 1) step of high concentration culture of lactic acid bacteria 3) 2) micro-immobilized lactic acid bacteria using sodium-alginate Making; And 4) preparing a printing paste using the microimmobilized particles of 3).

The fine immobilization may be widely used without limitation the immobilization method known in the art, preferably spray coagulation method or emulsion coagulation method may be used. The microbial immobilization method increases the survival rate of microorganisms and in a form that is inexpensively available, lactic acid bacteria can be protected from extreme environments such as freezing, high acid environment, high oxygen saturation and refrigerated storage. Therefore, the microimmobilization method has high stability even when stored at a low temperature, and can be used for the production of microbial TTI containing lactic acid bacteria that exhibit high metabolite productivity compared to the cells without immobilization. In addition, by using cryoprotectants such as glycerol, the survival rate of the immobilized cells in the lyophilization or drying process can be further increased.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 . Materials and methods

1.1 Strains and Passage Cultures

Lactobacillus , the strain used in this experiment amylovorus KCCM 40431 was distributed by the Korea Center for Microbiological Conservation. After incubation in de Man Rogosa Sharpe (MRS; Difo, Detroit, MI, USA) broth, the culture was dispensed in 1 ml of 15% glycerol and stored frozen at -80 ° C. The storage strain was used for the experiment by subcultured twice.

1.2 CaCO ₃ - Alginate Bead  Manufacture and high concentration culture

CaCO ₃ -alginate beads were dissolved in distilled water so that sodium alginate (Sigma Chemical Co., St. Louis, MO, USA) was 2% (w / v) and dissolved sufficiently using a stirrer, followed by 20% CaCO ₃ It was added to the mixture and stirred until it was sufficiently mixed. The sufficiently mixed mixed colloidal solution was added dropwise to a 0.1 M CaCl ₂ solution with a syringe (Gauge 18) while stirring at 200 ± 10 rpm to prepare CaCO ₃ -alginate, which was left for about 1 hour to strengthen beads. After washing three times with distilled water it was used sterilized in autoclave. L. amylovorus KCCM 40431 was inoculated in MRS broth with 20% CaCO ₃ -alginate beads (w / v) and incubated anaerobicly at 37 ° C. for 24 hours. The number of viable cells in the culture solution was diluted 10-fold and the number of viable cells was measured according to the standard plate counting method using BCP agar (Eiken Chemical Co., Tokyo, Japan) medium. In addition, the cells obtained by centrifugation (1,600 g for 20 min, 4 ° C) were washed twice with 0.1% (w / v) sterile peptone water and used for microimmobilization.

1.3 Fine Immobilization of Lactic Acid Bacteria

Microimmobilization of Lactic Acid Bacteria is Lee and Heo (Lee, KY, & Heo, TR (2000) .Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution. Applied and Environmental Microbiology, 66 (2), 869-873 ) and the like Brachkova. (See: Brachkova, MI, Duarte, MA , & Pinto, JF (2010) Preservation of viability and antibacterial activity of Lactobacillus spp in calcium alginate beads European Journal of Pharmaceutical Sciences , 41 (5), 589-596) was slightly modified to proceed with an electrospray gun (LPH-50-062G, Anest Iwata, Fukushima, Japan). Three emulsions were prepared by mixing glycerol and sodium alginate for fine immobilization. The mixing ratio of the emulsion is shown in Table 1. Centrifugal bacteria were inoculated into the emulsion at the level of 9.63 log CFU / ml and aseptically sprayed into 0.1 M CaCl 2 solution through a 0.6 mm nozzle with 98 psi compressed air using an electrospray gun. 0.1M CaCl2 solution was stirred at 200rpm for 1 hour to make almost perfect spherical fine particles, and the recovered immobilized lactic acid bacteria microparticles were washed twice with 0.1% (w / v) peptone water and used in subsequent experiments.

Ingredients Emulsion 1 Emulsion 2 Emulsion 3 Sodium alginate 1.5% (w / v) 2.0 g (w / v) 2.5 g (w / v) Glycerol ^a 10% (v / v) 10% (v / v) 10% (v / v) ^a The glycerol acted as cryoprotectant

1.4 Quantitative Analysis of Immobilized Lactic Acid Bacteria Fine Particles

Quantitative analysis of the immobilized lactobacillus microparticles was performed using the following methods. The lactic acid bacteria count inside the fine particles was shaken for 1 hour using 0.1 M phosphoric acid solution to destroy the gel structure, and diluted by 10-fold dilution method, followed by BCP agar (Eiken Chemical Co., Tokyo , Japan) was used to measure the viable cell number according to the standard plate count method.

After 1 g of immobilized lactic acid microparticles were suspended in 9 ml of 0.1% peptone water, an optical microscope (ECLIPSE E200, Nikon, Tokyo, Japan) and a scanning electron microscope (S-3000N, Hitachi, Tokyo, Japan) were used. The shape was observed. The number of fine particles was measured using a Petroff-Hausser counter (Electron Microscopy Sciences, Hatfield, PA, USA). The size and size distribution of the fine particles were measured by dynamic light scattering (DLS) using a BI-200SM goniometer (Brookhaven Instruments Corporation, Holtsvile, NY, USA) equipped with a BI-9000AT autocorrelator. At least 300 fine particles were used. The moisture content of the fine particles is 105 After drying for 24 hours at ℃, it was determined by the weight difference.

1.5. Water soluble printing paste manufacturer

10% (w / v) immobilized lactobacillus microparticles, 5.5% (w / v) nutrient, 10% (v / v) mixed indicator solution (mixed indicator solution contains 954% (w / v) bromocresol purple % (v / v) ethyl alcohol, 95% (v / v) containing 0.05% (w / v) bromocresol green and 95% (v / v) containing 0.1% (w / v) methyl orange Ethyl alcohol was prepared by mixing in a ratio of 1: 1: 1), 10% (v / v) glycerol and 5, 10, 15% (w / v) pullulan (pulluran, Hayashibara Shoji Inc., Tokyo, Japan) was mixed to prepare a 100 ml printing paste. The printing paste was homogenized at 7200 rpm for 30 minutes using a homogenizer, and the degree of suspension was checked under a microscope.

1.6. Water soluble printing paste evaluation

The drying properties of the paste were measured by thermogravimetric analyzer (Pyris 1 TGA, Perkin Elmer Inc., Waltham, MA, USA) at a constant temperature with time change of moisture content. 20 μl paste was taken in a sample container and dried in the steps shown in Table 2 to measure the weight change. The temperature of the sample was maintained at 42 ± 1 ° C. during drying. The wettability of the paste for pearl coated paper (Curious matallicsⓒ (pearl contents: 170g / m2), Samwonpaper Ltd., Seoul, Korea) was measured using a contact drop angle (DSA100, Kruss, Hamburg, Germany). contact angle). 1 ml paste was added to the syringe attached to the machine, and the contact angle was measured by dropping 2 μl drops of needle and pearl coated paper at a rate of 1.75 μl / s at a distance of 0.2 mm. The contact angle was accurately calculated by the Young-Laplace equation with the DSA100 applied from the drop contour at the contact point.

Step Heating condition First holding 1.0 min at 20 ℃ Heating From 20 ℃ to 50 ℃ at 10 ℃ / min Second holding 30 min at 50 ℃

1.7 Print and Print Evaluation

The print paste was printed through a silkscreen press. At this time, the strap strength of the silk cloth having 350 mesh was strapped to a tension value of 20 N / mm. Before printing, the silk plate was thoroughly dusted off, the paste was mixed well, and the pressure, angle and speed of the squeegee were optimized so that the thickness of the printed layer was 30 μm. The printed matter was dried at 40 ° C. for 10 minutes, and then activated in a dataator whose water activity was maintained at 0.98 to observe color change. The equilibrium water content of the printing paste was measured by a water activity meter (Thermoconstanter TH200, Novasina, Lachen, Swetzerland). That is, 1.0 g of a dry paste was placed in a chamber, and the water activity inside the device was maintained at 1.0 using distilled water, and then absorbed at equilibrium with a given water activity at a temperature of 25 ° C. to obtain an equilibrium water content.

The visibility of printed matter was evaluated by visual inspection and photographing. The photograph was taken with a Canon EOS 450D digital camera (Canon EOS 450D with 12.2 Mega Pixels, Tokyo, Japan) in a wooden box (50 × 50 × 60 cm) under dark conditions to prevent color rendering due to ambient light sources. At this time, the photographing light source was Natural Daylight (Philips, China) and a standard light source 6000K was applied. The distance between the printed matter and the digital camera is 25 cm, and the illumination angle is set to 45 degrees for the best lighting efficiency. The lens aperture is f = 4.5, the shutter speed is 1 / 125s, The photographs were taken at the maximum resolution (4272 x 2848 pixels). The captured images were saved as JPEG files and transferred to Apple computers (MacBook MC234KH / A, Cupertino, CA, USA).

1.8 Statistical Processing

All experiments were repeated three times, and each experiment was performed twice. Experimental results were calculated using the mean and standard deviation of each experimental group using SAS 9.1 for window (SAS Institute Inc., Cary, NC, USA) .The comparison between the experimental groups was analyzed by analysis of variance. ANOVA) was used. After the analysis of variance, the items with significant differences were tested by Duncan's multiple range test at the p <0.05 level.

Example  2

 2.1 Ca - To alginate  Preparation and Characterization of Immobilized Lactobacillus Microparticles

In order to develop an efficient mass production method of lactic acid bacteria, a CaCO ₃ -alginate bead system was applied to high concentration culture of lactic acid bacteria. When applying this system, more than 9 log CFU / ml lactic acid bacteria could be obtained. After 24 hours of incubation, L. amylovorus KCCM 4031 was incubated to 9.62 log CFU / ml. This level was about 15 times higher than without the CaCO ₃ -alginate bead system. These results indicate that the CaCO ₃ -alginate bead system is a very useful method for high concentration batch culture of lactic acid bacteria.

Microparticles prepared as shown in Figure 1 and Table 3 were spherical or elliptical shape.

The size and surface morphology of the immobilized lactobacillus microparticles was dependent on the composition of the colloidal solution. As the wet weight of the microparticles increases, the dry weight also increases, indicating that the prepared microparticles are particles homogeneously distributed in alginate, lactic acid bacteria, water, and glycerol. The number of lactic acid bacteria immobilized on the immobilized lactic acid microparticles was 8.77 ± 0.13 log CFU / g, 8.97 ± 0.03 log CFU / g, and 8.56 ± 0.15 log CFU / g, respectively. In terms of immobilization efficiency, these were 91.06 ± 1.29%, 93.09 ± 0.26%, and 88.81 ± 1.58%, respectively. The size of microparticles tended to decrease as the concentration of alginate increased, and the average diameter was found to be 1670.7 ± 148.0 ~ 2930.8 ± 305.7 nm. In the case of 2.0 LCAMs, the number of particles was the highest (86.99 ± 1.89) × 106 particles / g, followed by 2.5 LCAMs, and the number of particles was (53.33 ± 1.23) × 106 particles / g. The surface area of the microparticles was calculated using the average diameter and ranged from 280.78 ± 58.18mm² / g to 1126.47 ± 226.52 mm² / g. The surface area of 2.0 LCAMs was 1126.47 ± 226.52 mm² / g, which is about 4 times larger than that of 1.5 LCAMs. However, 2.5 LCAMs had a surface area larger than 1.5 LCAMs but the lowest immobilization efficiency. This was thought to be because 2.5 LCAMs had the smallest particle size and did not have enough space to fix the lactic acid bacteria.

Microparticles Encapsulation
efficiency (%) Moisture content
(%) Average diameter ^d)
(nm) Numbers ^e)
(10 ⁶ particles / g) Surface Area ^f)
(mm ² / g) 1.5 LCAMs ^a) 91.06 ± 1.29 86.269 ± 0.024 2930.8 ± 305.7 10.33 ± 4.19 280.78 ± 58.18 2.0 LCAMs ^b) 93.09 ± 0.26 85.451 ± 0.012 2003.2 ± 202.7 86.99 ± 1.89 1126.47 ± 226.52 2.5 LCAMs ^c) 88.81 ± 1.58 84.395 ± 0.011 1670.7 ± 148.0 53.33 ± 1.23 470.09 ± 82.88 ^a) Lactic acid bacteria loaded Ca-alginate microparticles containing 1.5% (w / v) sodium alginate
^b) Lactic acid bacteria loaded Ca-alginate microparticles containing 2.0% (w / v) sodium alginate
^c) Lactic acid bacteria loaded Ca-alginate microparticles containing 2.5% (w / v) sodium alginate
^d) The size of microparticles was measured by dynamic light scattering.
^e) The number of microparticles per gram was counted using a Petroff-Hausser Counting Chamber.
^f) Surface area of microparticles per gram was calculated based on the average diameter and numbers.

Among the three immobilized lactic acid bacteria microparticles prepared in the present invention, the microparticles obtained by spray coagulation using 2.0% (w / v) alginate were confirmed to be particles having a uniform surface having an average diameter of 2930.8 ± 305.7 nm. It was.

1 is a view of the immobilized lactic acid bacteria microparticles observed using an optical microscope and a scanning electron microscope. Microparticles immobilized with different amounts of lactic acid bacteria inside the microparticles were obtained using an electrospray gun. However, the shape of the microparticles was not uniform. As shown in FIG. 2C, a non-uniform shape was formed due to the loss of a portion of the immobilized lactic acid bacteria microparticles. This phenomenon has been determined that the alginate has not been in a CaCl ₂ that acts as a cross-linking agent in combination with Ca ₂ ⁺ promote the Ca ₂ ⁺ diffusion by sufficient stirring to form a gel occurs. In addition, the microparticles that did not immobilize the lactic acid bacteria could be observed. Production of microparticles not immobilized with lactic acid bacteria could be suppressed by increasing the concentration of lactic acid bacteria in the emulsion shown in Table 1. Based on the results, in order to obtain uniform microparticles, 2.0% (w / v) of alginate was used for lactic acid microimmobilization .

2.2. Features of Water-based Printing Paste

Aqueous printing pastes consisted of three important components. First, the immobilized lactic acid microparticles that induce a gradual color change of the pH indicator, the nutrients that can produce lactic acid, the mixed indicator solution that visualizes acidification by the resulting lactic acid, and the dispersant and aqueous printing paste of the immobilized lactic acid microparticles. It is a pullulan (pullulan, which acts as a texturing agent) to give the desired physical properties as a binder. These components were confirmed to be screen printed on the polymer material in the desired shape.

2.2.1 Printing Paste Contact angle

θ (Deg) Substrate 1.5 LCAMs ^d) 2.0 LCAMs ^e) 2.5 LCAMs ^f) 5% Pullulan ^a) 64.8 ± 3.6 68.0 ± 1.7 64.8 ± 6.6 10% Pullulan ^b) 80.4 ± 1.1 68.8 ± 3.0 77.6 ± 2.1 15% Pullulan ^c) 86.4 ± 1.7 67.0 ± 1.7 85.2 ± 1.2 ^a) Water based printable paste containing 5% (w / v) pullulan.
^b) Water based printable paste containing 10% (w / v) pullulan.
^c) Water based printable paste containing 15% (w / v) pullulan.
^d) Add the lactic acid bacteria loaded Ca-alginate microparticles containing 2.5% (w / v) sodium alginate
^e) Add the lactic acid bacteria loaded Ca-alginate microparticles containing 2.5% (w / v) sodium alginate
^f) Add the lactic acid bacteria loaded Ca-alginate microparticles containing 2.5% (w / v) sodium alginate

The contact angle was measured using a pendant drop tensiometer to evaluate the printability of pearl coated paper of aqueous printing paste containing 5, 10 and 15% of pullulan, respectively. The contact angle was measured to be less than 90 ° at all concentrations, but these values varied with the concentration of the pastes. Table 4 shows the contact angle values initially measured when the paste was added to the film. As the concentration of pullulan increased, the contact angle tended to increase. However, in the case of the Pluran-2.0% LCAMs system, this tendency was deviated. The highest contact angle was measured due to high interfacial tension and poor wettability in the 15% Pulran-1.5% LCAMs system. It was thought that high interfacial tension was generated due to the increase in viscosity due to the high concentration of pullulan.

The Pluran-2.0% LCAMs system exhibited a moderate contact angle (about 68 °), while the 15% Pulran-1.5% LCAMs system had the highest contact angle (about 86 °). Contact angles are frequently used as experimental indicators of interfacial tension and wettability. In general, when the contact angle is smaller than 90 °, it can be said that the surface state is close to hydrophilic, and when it is larger, it is said to be hydrophobic. When the contact angle exceeds 160 °, the surface state is expressed as superhydrophobic. Because of the contact angle of water, the Pluran-2.0% LCAMs had adequate wettability with respect to pearl coated paper.

2.2.2 Drying Properties of Printing Pastes

Such aqueous printing paste must be dried to form the desired shape on the surface of the polymeric material. Since the paste has a drying property determined by the components as well as the manufacturing method, the drying property of the paste was evaluated by using a thermogravimetric analysis method.

Figure 2 shows the weight loss and weight loss rate when drying the pullulan-LCAMs system while heating at a rate of 10 ℃ / min. The Pluran-LCAMs system showed a distinct three stage drying process. In the first stage of increasing temperature from 15 ℃ to 20 ℃, a small amount of weight change was observed, and the rate of weight loss tended to increase slightly. This is believed to be due to the decrease in moisture. In the second stage, where the temperature was increased from 20 ° C to 50 ° C, a significant amount of weight loss was observed, which is considered the main drying process. Most of the water was removed at this stage (approximately 90%). In step 3 of maintaining the temperature of 50 ℃, there was no weight loss, so it could be confirmed that all moisture that could be removed was removed from the printing paste.

The weight loss rate during the drying process was higher in the second stage than the other two stages, and the weight loss due to the removal of most moisture occurred in the range of 20 ℃ to 50 ℃. In addition, the drying rate tended to increase as the concentration of pullulan increased. The drying properties of the paste obtained through thermogravimetric analysis were very useful for understanding the drying process of the printed matter and could also be applied to the inert process of microbial TTI. In general, high concentrations of texturing agents slow the growth of microorganisms by slowing the growth of microorganisms within the microbial TTI. Therefore, the concentration of texture formers affects the color change of the indicator. Taken together, the results indicate that 10% of flulan is suitable for the production of microbial TTI.

2.3. prototype  Microbial TTI Features of

After printing, the microbial TTI undergoes two moisture treatments. The first is the drying process for inactivation, and the other is to absorb moisture for activation before adhering to the product. Water absorption characteristics of the microbial TTI are shown in FIG. 3.

All samples showed two levels of hygroscopic curves. In the early stages, rapid moisture absorption occurred, followed by gentle moisture absorption. The moisture absorption rate was dependent on the concentration of pullulan. The higher the concentration of pullulan, the higher the moisture absorption rate. At the initial stage of absorption, higher moisture absorption rates were observed in 10% Pulran-2.0% LCAMs and 15% Pulran-2.0% LCAMs compared to 5% Pulran-2.0% LCAMs. Moisture absorption of all samples increased until equilibrium. For 10% Fulan-2.0% LCAMs and 15% Fulan-2.0% LCAMs, the water activity was less than 0.60 where microorganisms could not grow.

Microbial TTI contains microorganisms which grow depending on the storage temperature and acidify the medium. This acidification should begin at the beginning of the shelf life of the product. Therefore, these indicators must be inactive when not attached to the product and must be activated when attached to the product. Water must be supplied in the medium to activate the indicator. Accordingly, it was confirmed that 10% Pluran-2.0% LCAMs and 15% Pluran-2.0% LCAMs had adequate moisture hygroscopicity for microbial TTI development.

The visibility of the microbial TTI was evaluated visually and photographically. The most important capability of a digital camera is the ability to image not only the entire surface of the indicator, but only certain parts of interest. 4 shows that only a part can be selected to know the L a b value of a particular part of the TTI. In the present invention, when the fixed lactic acid bacteria grow in the water-soluble printing paste, the color of the microbial TTI changed from light green to colorless due to the pH change. In FIG. 4, it is considered that a small portion of the greenish green color selected from the indicator which was enlarged as much as possible did not use immobilized lactic acid bacteria, and the pH did not change uniformly on the indicator surface. After two days, the L value of the small lime green part selected was less than the L value of the surface as magnified as possible (68 vs. 88). Moreover, the a and b values were also low because the indicators tend to change to green and yellow. The visibility of the microbial TTI was excellent where the lactic acid produced by the immobilized lactic acid microparticles was used.

Claims (9)

Print paste for microbial TTI, characterized in that it contains lactic acid bacteria microimmobilized particles immobilized in sodium alginate of 1.5 to 2.5% (w / v).
The printing paste for microbial TTI according to claim 1, wherein the printing paste contains 10% (w / v) lactic acid bacteria microimmobilized particles based on the amount of the paste.
According to claim 1, wherein the printing paste for microbial TTI characterized in that it further comprises a mixed indicator solution, glycerol, nutrients, pulluran (cryluprotectant), cryoprotectant, cryostabilizer Printing paste.
The printing paste for microbial TTI according to claim 3, wherein the pullulan contains 5 to 15% (w / v) based on the printing paste.
Microbial TTI comprising the printing paste of any one of claims 1 to 4.
1) preparing CaCO 3 -alginate beads for high concentration culture;
2) inoculating the lactic acid bacteria in the medium containing the beads of 1) to cultivate a high concentration of lactic acid bacteria;
3) separating and recovering the lactic acid bacteria of 2);
4) separating and recovering the lactic acid bacteria of 3) using sodium alginate to fine fix; And
5) preparing a printing paste containing finely immobilized particles of 4).
7. The method of claim 6, wherein the microimmobilization is spray coagulation.
7. The method of claim 6, wherein the microimmobilization is an emulsion coagulation method.
A microbial TTI manufacturing method comprising using a microbial TTI printing paste prepared by the method of claim 6.

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