DESC:FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION: “METHOD AND SYSTEM TO CHECK BACTERIAL LOAD OF MILK BY METHYLENE BLUE DYE REDUCTION TEST”

2. APPLICANT:

(a) NAME : PROMPT EQUIPMENTS PVT. LTD.
(b) NATIONALITY : INDIAN
(c) ADDRESS : 3-B, VARDAN EXCLUSIVE,
NEAR STADIUM PETROL PUMP,
NEXT TO VIMAL HOUSE,
NAVRANGPURA, AHMEDABAD-380014,
GUJARAT, INDIA

3. PREMABLE TO THE DESCRIPTION:
PROVISIONAL

The following specification describes the invention. ?COMPLETE

The following specification particularly describes the invention and the manner in which it is to be performed.

FIELD OF THE INVENTION:-

The present invention relates to method and system to check bacterial load of milk by methylene blue dye reduction test. More particularly, the method and system provides an automated and digitized solution to eliminate manual intervention and enhance the accuracy, repeatability and efficiency of the MBRT process. The method and system further facilitates real-time monitoring, data transmission and shelf-life prediction.

BACKGROUND OF THE INVENTION:-

In today’s dairy industry, maintaining the microbiological quality of milk is more critical than ever, as consumer demand for safe and fresh dairy products continues to rise. With increasing milk production volumes and stricter regulatory standards, the need for fast, accurate, and efficient bacterial load testing has become paramount. Conventional method like the Methylene Blue Reduction Test is widely used due to the simplicity and cost-effectiveness.

Methylene Blue Dye Reduction Test, commonly known as MBRT is used as a known method to assess the microbiological quality of the raw and pasteurized milk. Methylene Blue Dye Reduction test (MBRT) is used to analyze milk based on microbiological quality of the milk. A methylene blue dye is added to the milk and its rate of discoloration defines the microbiological quality of the milk. The MBRT is one of the critical test to check bacterial load of milk and hence a most important for the dairy industry.
Methylene Blue Reduction Test (MBRT) helps in evaluating the contaminant factors as involved due to the presence of microbes in the milk.

The Methylene Blue Reduction Test (MBRT) is based on the fact that the color imparted to milk by the addition of a dye such as methylene blue disappears more or less quickly. The removal of the oxygen from milk and the formation of reducing substances during bacterial metabolism cause the color to disappear.

The larger the numbers of bacteria in the milk, sooner the change in color take place. Hence, use of methylene blue reduction test becomes a valuable test for determining the relative number of bacteria present in a milk samples.

In a conventional process,a known quantity of the dye is added to a known quantity of milk in a test tube, and the tubes are held in a water bath at 37ºC. The tubes are checked for change in the color of the milk from Blue to original milk color. The time required to change this color indicates the level of contamination. More the time indicates a better quality of the milk. A good quality of milk would have over 5 hours of MBRT time.

Patent application number 660/DEL/2013 describes a strip-based testing kit for identifying milk adulteration and assessing microbial freshness. The strips are pre-treated with specific reagents tailored to detect various adulterants and microbial activity, producing a visible color change in the strip or milk sample upon contact. However, it does not disclose the real time bacterial load analysis through an automated MBRT-based system which provides accurate digital results, remote monitoring, and real-time data access which minimizes human involvement.

Patent application US20140287428A1 discloses a method for detecting bacteria in milk especially mastitis-causing types by using stained antibodies that bind specifically to target bacteria. It allows simultaneous detection of multiple bacterial types through color changes or concentration differences. The method may include preprocessing steps like protein or fat removal to improve clarity and detection accuracy and is tailored for use in milk analysis systems. However, it does not involve a specific automated system based on the Methylene Blue Reduction Test (MBRT) for checking the bacterial load in milk.

Further, in a conventional approach of MBRT, a person needs to manually observe the color of the milk in the test tube at regular intervals to note the readings and infer results. The test results are affected by the quantity of the dye, the quantity of the milk and temperature of the water bath and regular interval, 30 minutes inversion of the test tube. Any deviations from the required quantity have a major impact on the accuracy of the results. Since all the steps in conventional processes are performed manually, the test is normally taken as an indicative test rather than a definitive test.The conventional process does have deficiencies and drawbacks during the whole process of MBRT test.

Following are the drawbacks of the conventional process of Methylene Blue Reduction Test (MBRT) of the Milk:
• Strong possibility of the deviation from required results during conventional method with human intervention or manual process.
• Any deviations in volume of milk and Methylene Blue had a major impact on the accuracy of the results. Every 1ml increase in milk volume (by keeping MB volume 1ml) will reduce MBRT by 2-6 %, while very 1ml increase in Methylene Blue volume (by keeping milk volume 10ml) will increase MBRT by 15-20 %.
• Possibility of improper mixing of the dye and milk.
• High power consumption as complete water bath is required to be maintained at desired temperature, even for single sample.
• In conventional method manual process required to note the time taken to reduce the color.
• A long time, which is exactly equal to original shelf life of milk is required to know the final results.

Hence, the above mentioned technical problem is solved by the present invention which introduced method and system to check bacterial load of milk by methylene blue dye reduction test. It tackles the limitations of traditional approaches by incorporating modern technologies such as automation and digitization into the MBRT process. Also, the method for execution of MBRT via automation and digitization which is accurate fine gained control on the entire unit to ensure perfect execution of the whole process, reliable and have ability to get accurate result in short period of time.

OBJECT OF THE INVENTION:-

The principal object of the present invention is to develop method and system to check bacterial load of milk by methylene blue dye reduction test.
Further object of the present invention is to develop a method and system to digitize the process to check bacterial load of milk with quick estimation of the MBRT by using predictive model with utmost efficacy & precision.

One more object of the present invention is to develop a method and system thereof to control the variables of the process like milk & dye volume, time, temperature, inversion at regular interval and an exact decolorization time.

Further object of the present invention is to develop a method and system to check bacterial load of milk which eliminates the possibility of improper mixing of the dye and milk.

Another object of the present invention is to develop a method and system to check bacterial load of milk which reduces power consumption, time to monitor the color change during the process and optimally reduces a long time required to get the final results.

One more object of the invention is to develop a method and system to check bacterial load of milk with accurate and low-cost heating mechanism with capacity to heat and maintain the temperature of each tube accurately.

Further object of the present invention is to develop a method and system to check bacterial load of milk has capability to predict the shelf life of milk before the actual shelf life of milk.

Yet another object of the present invention is to provide a method and system to check bacterial load of milk with a high-accuracy predictive model and to enable the entire data available on a central portal for management, reporting and control.

Yet another object of the present invention is to provide method and system to check bacterial load of milk, which is free from the intentional or unintentional error in MBRT time arise due to improper volume of Milk / Methylene Blue dye concentration.

Still, another object of the present invention is to provide a method and system to check bacterial load of milk to get accurate result in short period of time, thereby enabling early conclusiveness leading to enhanced profits.

Yet another object of the present invention is to provide a method and system to check bacterial load of milk, which is capable of showing progress of test on the display device of system without taking test tube out from test cavity.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the present embodiment when taken in conjunction with the accompanying drawings.

Figure 1 represents block diagram of the present invention.

SUMMARY OF THE INVENTION:-

The present invention relates to method and system to check bacterial load of milk by methylene blue dye reduction test. The present invention particularly relates to an automated system with predictive model to check bacterial load of milk based by Methylene Blue Reduction Test (MBRT) comprises plurality of sensors, a microcontroller, a motor & gear system, a battery & charging system, a display device, a real time clock and wireless communication device. The system enables the entire data available on a central portal for management, reporting & control. The entire system is been made remote controlled operational over the internet. The system enables digitization of the MBRT information and data analysis through mobile application and synchronies over the internet. This automated system eliminates human intervention for MBRT time with high-accuracy & precision.

DETAIL DESCRIPTION OF THE INVENTION:
Before explaining the present invention in detail, it is to be understood that the invention is not limited in its application to the details. The invention is capable of other embodiment and of being practiced or carried out in a variety of ways. It is to be understood that the phraseology and terminology employed herein is for the purpose of description and not of limitation.
It is to be also understood that the term “comprises” and grammatical equivalents thereof are used herein to mean that other components, ingredients, steps, etc. are optionally present. For example, an article “comprising” or “which comprises” components A, B, and C can consist of (i.e. contain only) components A, B and C or can contain not only components A, B and C but also contain one or more other components.
The invention is capable of other embodiments, as depicted in different figures as described herein and of being practiced or carried out in a variety of ways. It is to be understood that the phraseology and terminology employed herein is for the purpose of description and not of any limitation.

Before discussing specific embodiments, it is to be noted that the term “user device” may include mobile phone, tablet, desktop and laptop.

The “microcontroller” is a compact integrated circuit designed to govern a specific operation in an embedded system. It can include a processor, memory and input/output peripherals on a single chip. It mostly used in devices, robots, home appliances and motor vehicles etc.
The present invention relates to method and system for methylene blue dye reduction test to check bacterial load of milk comprises a heating block with a temperature sensor (6) configured to maintain the temperature during the MBRT with capacity to accommodate more than one test tube, each test tube is uniquely identified by a Quick Response (QR) code and an internal channel code. A wide band chromatic sensor (5) is used to confirm the volume of Milk / Methylene blue filled in the test tube up to the desired volume. It also continuously monitors the changes in colour and also estimates the status of the test. Whereas the volume of methylene blue at a concentration of 1 ml is significant for quality control and detecting this concentration accurately is crucial. A microcontroller (1) is configured to manage the whole system automatically through sensors which also control the addition of required quantity of the dye. A motor & gear module (7) which is configured with the heating block (6) to thoroughly mix the dye and milk by facilitating the inversion at an angel of 100-120 degree from the normal position for three times to facilitate mixing of dye and milk at regular interval of 30 min or so.

In preferred embodiments of the present invention further includes the digital display device (4) configured to visually indicate the display of the data during the test. Further, it is cloud based system that provides a real time data monitoring and a process control through user device to ensure the perfect execution of the process. In preferred embodiment of the present invention RTC (real time clock)(2) is configured to obtained precise time and the system is accommodated with its own battery backup system (8) to ensure that the test is not interrupted due to power failure.
In the preferred embodiments, the microcontroller(1)is configured with each component peripherally to control an automatic process and transferred all notification/signal to the user device through wireless communication device(3).

In preferred embodiments, the automated system have a capacity to heat and maintain the temperature of each unique tube properly without affecting any other tube with predetermined temperature set by the user device to heat the container uniformly.

Method and system to check bacterial load of milk by methylene blue dye reduction test comprises following steps:

a) 10 ml of milk and 1ml of methylene blue dye are dispensed into a test tube and placed into the heating block (6) maintained at 37°±0.5°C, whereas the temperature monitored by a sensor (6) that transmits data to the microcontroller (1).
b) The volumetric estimation of Milk / methylene blue dye in the test tube is performed by the wide band chromatic sensor (5) and microcontroller (1) and transmits the captured data to the microcontroller for further processing.

c) After estimation of the volume by wide band chromatic sensor (5), the time of keeping the test tube in the heating block(6) is registered digitally as the starting time with the help of Real Time Clock(2),

d) The test is monitored by wide band chromatic sensor (5) at regular interval of 1 min,

e) The rotation of the heating block (6) is facilitated by a motor and gear system (7), which tilts it at an angle of 100–120° every 30 min to ensure proper inversion and mixing of the methylene blue dye and milk inside the test tube.

f) The time taken to reach the end point of the MBRT test is automatically recorded by the system via the microcontroller (1) at the moment when the dye stops showing its color, marking the completion of the test.

g) The difference between the start time and end time is recorded as the MBRT time (shelf life of milk), which is stored and transmitted to the user device via the wireless communication system (3), while all related notifications and test data are also imparted through the predictive module and visually presented on the digital display device (4).

It is to be understood that in step (a), the heating block with temperature sensor (6) has a capacity to accommodate more than one test tube at a time.

It is to be understood that in step (a), the precise volumes of methylene blue is critical to ensure the accuracy, repeatability, and reliability of the automated MBRT process. Any deviation can affect the reaction kinetics and lead to incorrect interpretation of bacterial load.

It is to be also understood that the wide band chromatic sensor (5) is configured for precise loading of the milk and methylene blue dye. Also, if the added dye volume deviates from the recommended value (1 ml), then wide band chromatic sensor (5) sent a notification to the operator/authority to discard the test. The data taken by the wide band chromatic sensor (5) has capability to predict shelf life milk sample before its actual shelf life.

The Microcontroller (1) is the central unit for receiving & updating the data from each component of the automated system via digital device with predictive model. The data of predicted shelf life sent to the operator/user device in the central laboratory with the help of wireless communication device (3).

The present invention is illustrated more in details in the following experimental example. The example describes and demonstrates the embodiments within the scope of the present invention. This example is given solely for the purpose of illustration and is not to be construed as limitations of the present invention, as many variations thereof are possible without departing from spirit and scope.
Example 1 illustrates that the system has capability to detect deviation in required volume (1ml) of Methylene Blue dye with accuracy of 80-90 %.

Several trails were conducted to determine whether the Methylene Blue dye in milk is 1?ml through wide band chromatic sensor (5) in conjunction with Machine Learning Techniques. The present invention aims to accurately differentiate between samples labeled as Class 1 (1 ml) and Class 2 (other concentrations).

The trails were conducted using different Milk types including Pasteurized Buffalo Milk, Pasteurized Cow Milk, Pasteurized Homogenized A2 Cow Deshi Milk, Pasteurized Taaza Tonned Milk, Pasteurized Tea Special Milk and Raw Cow Milk.

Wide band chromatic sensor (5) was used to collect data from multiple milk types. Each sample was measured under controlled conditions to ensure consistency. Also each milk type was analyzed using the same sample size to maintain uniformity across the experiment.

It is to be understood that predictive model was trained using labeled data, with Class 1 representing samples with 1 ml of Methylene Blue and Class 2 representing other concentrations of Methylene Blue.

It is to be understood that the classifier includes but not limited to “Random Forest classifier” and it was due to its robustness and ability to handle complex data.

It is to be understood that the hyperparameter tuning was performed to optimize accuracy and minimize errors.

Here, the accuracy defines the percentage of correctly classified samples, whereas the F1 Score defines balance between precision and recall, important for evaluating binary classification performance.

Table 1 shows different milk type, accuracy (%) and F1 Score (%):

Sr. No Milk type Accuracy (%) F1 Score (%)
1 Pasteurized Buffalo Milk 82.38 84
2 Pasteurized Cow Milk 86.88 87
3 Pasteurized Homogenized A2 Cow Deshi Milk 87.03 88
4 Pasteurized Taaza Tonned Milk 81.31 81
5 Pasteurized Tea Special Milk 83.87 84
6 Raw Cow Milk 91.43 92

Observation and conclusion:
From aforementioned results, it is observed and concluded that the presently invented method identifies whether Methylene Blue concentration in milk is 1 ml with an overall accuracy of approximately 85.48%. The system shows potential for practical applications in milk quality testing, offering reliable performance across several milk varieties.

Example 2 illustrates the correlation between MBRT time with different volumes of milk and methylene blue solution.

Raw milk samples were collected. The collected samples were mix thoroughly. These milk samples were added in test tubes at different volumes. Further, different volumes of methylene blue solution were added in test tubes. All test tubes were closed with stoppers and were inverted for mixing. The test tubes were kept in water bath at 37?C. The initial time of MBRT test was noted. The color changes were observed from blue to decolorized and decolorized time of MBRT test was noted down. MBRT time was calculated by taking difference between decolorized time and initial time.

Increasing methylene blue or decreasing milk volume generally increases MBRT time, while increasing milk volume tends to reduce it. Small changes, like 0.5 ml, have minimal effect. Overall, MBRT is sensitive to both dye and milk quantities.

MBRT time (minutes)
Milk volume Methylene blue solution Sample 1 (13/03/2024) Sample 2 (14/03/2024) Sample
3 (18/03/2024) Sample
4 (19/03/2024) Sample 5 (28/03/2024) Sample 6 (28/03/2024)
10 ml 1 ml 300 250 270 120 245 295
10.5 ml 1 ml 300 250 265 120
11 ml 1 ml 285 255 255 120 225 290
11.5 ml 1 ml 285 255 255 120
12 ml 1 ml 285 260 250 120 225 285
12.5 ml 1 ml 280 260 250 120
13 ml 1 ml 270 240 250 120
9 ml 1 ml 315 260 280 120 255 300
9.5 ml 1 ml 315 255 275 130
10 ml 1.1 ml 310 255 275 130 250
10 ml 1.2 ml 320 270 280 135
10 ml 1.3 ml 320 270 280 135 270 300
10 ml 1.4 ml 320 275 285 135
10 ml 1.5 ml 325 280 290 140 275 315
10 ml 2 ml 345 300 310 150 285 330

Observation and conclusion:

From aforementioned results, it is observed and concluded that adding more than 1.0 ml of methylene blue solution to raw milk during the MBRT can alter the test outcome. An excess of 0.5 ml can increase the MBRT time by 15–20 minutes, while an additional 1.0 ml can extend it by 30–40 minutes. Similarly, increasing the milk volume by 2.0 ml can reduce the MBRT time by 10–20 minutes.

The present invention is innovative, smart and intelligent method and system for the MBRT which ensures that the accurate process is followed.

In accordance with one more aspect of the present invention of an automated system with predictive model and the method for MBRT of the milk does have multiple benefits such as:

• It eliminates human intervention and automates whole process to control the variables like milk & dye volume, time, temperature, inversion at regular interval and an exact decolorization time etc.
• The invented automatic system with predictive model via digital device and the method thereof for MBRT provides precise results with utmost precision and does have fine control on each step to ensure safety, efficacy & precise execution of the whole process.

• Each component of the present automated system and the method thereof for MBRT of the milk has been developed with high efficacy & precision to ensure effective quality control in the dairy industry.

• It enables to get accurate results via mobile application with quick estimation of the MBRT time using an automated system with predictive model thereby enabling early, better decision making and leading to enhanced profits.
List of Reference Numerals
(1) Microcontroller
(2) Real time clock
(3) Wireless Communication Device
(4) Display Device
(5) Wide band chromatic sensor
(6) Heating block with Temperature sensor
(7) Motor & Gear system
(8) Battery & Charging System

,CLAIMS:WE CLAIM:

1. A method to check bacterial load of milk by methylene blue dye reduction test, the method comprising:

a) dispensing and ensuring a predefined volume of milk and methylene blue dye into a test tube within a heating block(6) maintained at 37°C ± 0.5°C and transmitting the data to microcontroller (1);

b) performing the volumetric estimation of milk and methylene blue dye in the test tube, the wide band chromatic sensor (5) is electronically communicated with microcontroller (1), to transmit the captured data to the microcontroller for further processing;

c) recording the start time of the test using a Real-Time Clock (RTC) (2) once the sample is placed in the heating block(6), following the volumetric estimation completed by the wide band chromatic sensor (5);

d) monitoring the rate of change of dye concentration at predefined intervals through the wide band chromatic sensor (5);

e) tilting the heating block (6) at an angle of 100–120° by a motor and gear system (7) to ensure proper inversion and mixing of the methylene blue dye and milk inside the test tube;

f) detecting and recording the end time when the dye is fully decolorized by the system via the microcontroller (1), indicating the MBRT endpoint;

(g) recording and storing the difference between the start time and end time as the MBRT time (shelf life of milk) and transmitting the data to the user device via the wireless communication system (3), while simultaneously imparting all related notifications and test data through the predictive module and visually presenting them on the digital display device (4).

2. The method as claimed in claim 1 step (a), wherein the heating block (6) is capable of accommodating multiple test tubes while maintaining required temperature of each test tube.

3. The method of as claimed in claim 1 step (b), wherein wide band chromatic sensor (5)continuously monitors the color change and sends real-time updates to the microcontroller.

4. The method of as claimed in claim 1 step (b), wherein a predictive model analyzes data obtained by the wide band chromatic sensor (5) to estimate the shelf life of the milk before its actual shelf life.

5. A system to check bacterial load of milk by methylene blue dye reduction test comprises a microcontroller (1) plurality of sensors, a motor & gear system (7), a battery & charging system (8), a display device (4), a real time clock (2) and a wireless communication device (3);
a heating block (6) configured with the temperature sensor to maintained the required temperature throughout the test and accommodate a plurality of test tubes, each test tube uniquely identified by a Quick Response (QR) code and an internal channel code; wide band chromatic sensor (5) electronically communicated with themicrocontroller
(1) to confirm the volume of Milk / Methylene blue filled in the test tube up to the desired volume which continuously monitor color changes during the test anddetermine the test status based on the dye reduction rate;

said microcontroller (1) peripherally configured with and operatively connected to each component including the heating block (6), wide band chromatic sensor (5), motor & gear module (7), real-time clock (2), display device (4) and wireless communication device (3) to automatically control the entire MBRT process, manage sensor data, control the addition of methylene blue dye, and transmit notifications, alerts, and test results; a motor and gear module (7) operatively coupled to the heating block (6) and adapted to facilitate the inversion and thorough mixing of the milk and dye solution inside each test tube; the digital display device (4) configured to display real-time information including operational status, test progress and results;the wireless communication device (3) configured to transmit all data, notifications and signals from the microcontroller (1) to the user device, enabling real-time remote monitoring and control via a cloud-based interface; a RTC (real time clock) (2) is configured to obtained precise time and the system is accommodated with its own battery backup system (8) to ensure that the test is not interrupt due to power failure.
6. The method as claimed in claim 1, wherein the system improves MBRT testing by reducing manual errors, optimizing power consumption, and enabling faster decision-making in dairy quality control.

Dated this on April 21st, 2025