Authors: Ananya Garg, Pranava Jana, Soumya Rai, Jeslyn Wu
Research Advisor: Ms. Hiral Dantara
Peer Reviewer: Haania Mahmood
Professional Reviewer: Pascale S. Guiton
ABSTRACT
Milk is a substance with high nutritional value and is highly prone to microbial contamination. Expiration dates on milk aim to predict a date signifying when the milk can no longer be safely consumed due to chances of contamination. This study aims to identify any possible coliforms that may develop in milk before and after the expiration date. By performing tests such as Methylene Blue Reduction Test (MBRT) and looking for growth on selective media for microbial activity, we determined the effectiveness of pasteurization on milk quality until expiration. Due to the high viability of coliform growth in milk, various tests such as acid and gas production and IMViC tests were performed to isolate and identify any strains of coliforms which can cause disease. We tested milk samples at 3 intervals: 5 days before expiration, on expiration, and 5 days after expiration. We found no microbial presence except in one of three samples after expiration. We used reference charts/tables along with picture references of different bacteria strains growing on agar to use visual observations to cross-reference and determine that we did not isolate E. coli. The results show that expired milk is unsafe and is prone to microbial contamination after the date of expiry. Due to the heat-shock processing methods commonly used by milk manufacturers, milk expiration dates are displayed as a precaution for consumers’ well-being as the true-expiration date is often after the labeled date.
INTRODUCTION
Milk is a vital part of the human diet due to its extremely large nutritious value [1]. Fresh milk and dairy products are valuable sources of proteins, fat, and energy; thus making dairy an important part of daily meals [2]. As infants, milk generally constitutes the entire infants’ diet. Even children, youth, and adults drink milk for calcium, vitamins, and minerals [3]. It is incorporated into many dairy products, including cheeses, yogurts, and cakes. Plain milk is sold in bulk as well as smaller quantities like individual servings. Several different types of milk exist, such as milk with different amounts of fat, which can vary from nonfat milk to whole fat milk. Additionally, different sources of milk are known, such as cows, goats, camels, and sheep. In the modern world, milk can also be flavored, like strawberry-flavored milk, or chocolate-flavored milk. Milk can also be lactose-free, organic, pasteurized, or raw. Regardless of the type of milk or the species the milk comes from, fat, protein, lactose, and ash are the main components of milk [4]. These components make milk very susceptible to contamination. Products derived from raw and completely unprocessed milk, can harbor many microorganisms and be a source of foodborne pathogens which are a hazard to anyone who consumes the product [5]. Milk, in all its various forms, is used in the creation of dairy products, and therefore, the quality of such dairy products is based on the microbiological and bacterial quality of the milk from which it was derived.
As a basic standard that is almost always followed, expiration dates are found on most foods to signify when the product is prone to contamination and can no longer be safely consumed. Similarly, in milk, after this expiration date, milk may become prone to microorganisms, including bacteria, which are harmful to consume. Expiration dates for milk are often determined in mass and aim to broadly name a date after which the milk is not safe to use [6]. Different types of milk, such as raw milk and pasteurized milk, may expire sooner or later, relatively. Raw milk tends to have a larger amount of coliforms that are present due to poor hygiene, contaminated water, unsanitary milking practices, and improperly washed equipment and hands [7]. Studies have found that the average coliform counts in raw milk can range from 2.0×105 to 1.0×104 CFU/ml [8]. Due to the high bacterial and coliform presence, raw milk tends to spoil faster in comparison to pasteurized milk. However, pasteurized milk can also be easily contaminated, which can result in early spoilage of the milk.
By definition, coliforms are rod-shaped, gram-negative, non-spore-forming bacteria that have the capability to ferment lactose with the production of acid and gas within 48 hours at 32°C-35°C [9]. They are commonly used as indicator organisms to validate the quality of milk available in multiple parts of the world [10]. The presence of coliforms indicates fecal and/or environmental contamination [13]. As a result, the coliform standards are incorporated into a number of documents and regulations, including the U.S. Food and Drug Administration’s Grade “A” Pasteurized Milk Ordinance [11]. Bacteria that fall into the category of coliforms, mainly come from the family Enterobacteriaceae, which encompasses the Escherichia, Serratia, and Klebsiella species among others [9]. However, strains of Aeromonas hydrophila from the family of Aeromonadaceae have been identified as coliforms due to their ability to ferment lactose within 48 hours at 32°C-37°C, despite differing opinions on whether or not they should be considered a coliform[12].
Previous studies related to milk and coliform count have found post-processing contamination (PPC) with coliforms in 7.6%-26.6% of U.S. milk samples tested between the years 2001 and 2010 [13]. Pasteurized milk samples containing coliforms had significantly higher bacterial quantities and lower sensory scores for shelf-life compared to samples that were not contaminated [13]. Sensory scores are part of a product evaluation protocol and are used to analyse responses to products, in this case milk, through the five senses. The presence of coliform in the milk also indicates PPC of the product or pasteurization failure due to the bacteria’s ability to withstand extreme heat [10]. Many coliforms that are commonly found in milk are psychrotolerant, meaning that they can grow at high rates when refrigerated [14]. A recent study found that these coliform species showed the ability to grow substantially after 10 days of refrigeration, which led to bacterial production of lipolytic and proteolytic enzymes. These enzymes can cause flavor, odor defects, and abnormalities in milk [15], which make the presence of coliforms in milk detrimental to the quality of the milk, milk products, and consumer acceptance.
Coliforms can be identified by multiple tests including the Multiple Tube Fermentation Technique and Most Probable Number [16]. After the bacteria are confirmed to be a coliform, the Indole test, Methyl red test, Voges-Proskauer test, and Citrate test which uses agar plates and various other broths, can be performed to determine which specific type of bacteria is present in the sample. Bacterial colonies are counted on agar plates to determine their density in the sample. Several characteristics of bacterial colonies, including size, shape, and color, are often used to distinguish among bacteria that might be present in a particular sample. Additionally, more than one type of coliform may be present in the same milk sample.
The presence of coliforms such as Escherichia coli indicates the possibility that other microorganisms are present in the milk, if consumed, these microbes can be toxic and lead to a public health hazard. E. coli has been frequently used as an indicator of poor hygienic conditions in microbiological analyses of food, mainly due to its ubiquitous presence in food products generated under unsanitary conditions such as milk [1,17]. The consumption of food products contaminated with coliforms is likely to lead to infectious diseases, including urinary tract infections, pneumonia, septicemias, and soft tissue infections These microorganisms can also cause one of many severe pyogenic infections that have a high fatality rate if left untreated [18]. Furthermore, disease-causing coliforms can spread rapidly in a hospital environment and cause nosocomial outbreaks and other public health crises [19].
Since expiration dates are given on a bulk level, the true-expiration date for a carton of milk may vary significantly from the date printed on the carton. Going into this experiment, it was hypothesized that the pasteurization of milk significantly decreases the number of microorganisms present in a sample of milk. This study aimed to collect different milk samples and research how the milk quality is before expiration, the day of expiration, and directly after the expiration date.
METHODS
Sample Collection: All milk samples were pasteurized and processed milk brands commonly found in convenience stores and were chosen to be cow’s milk expiring around the same date. The three brands chosen for our experiments were Lucerne, Producers, and Organic Valley. While Lucerne and Producers were the experimental groups containing 2% milkfat each, Organic Valley was whole fat and unaltered milk fat.
Microbial Detection: In order to help identify any possible active or thriving microorganisms inside the samples of milk, a Methylene Blue Reductase Test was performed with 2mL of 0.01% Methylene Blue that was added to 10 ml of fully concentrated milk. Methylene Blue is an indicator that loses its strong distinctive blue color in the presence of oxygen, suggesting microbial activity inside sealed test tubes in the milk [20]. Based on the rate of color loss, one can determine whether the milk is contaminated with microorganisms or whether the color loss is from oxygen previously present in the test tubes (meaning there is no microbial activity).
Isolation of Microorganisms in the Milk Sample: Milk samples at 10-1 dilutions were streaked onto nutrient agar plates using inoculation loops and quadrant streaking. Agar streaking provides isolated bacteria colonies that may be in the milk [21]. In the case of bacterial growth, the colonies in the 4th quadrant were used to help perform subsequent testing in order to identify which strain of bacteria was isolated on all plates.
Coliform selection: Lauryl Tryptose Broth was used to help isolate the sugar fermenting bacterial colony and confirm if coliform growth was present. Milk was added to the LTB Durham medium to detect the presence of lactose-fermenting coliform using the accumulation of gas in the Durham tube as a proxy. [21]. Lauryl tryptose sulfate present in the broth is a selective agent that inhibits the growth of bacteria incapable of lactose fermentation, further distinguishing coliforms from other strains of bacteria that may be present [21].
Microbial Identification: The microbes were determined to be fungi or bacteria depending on the shape and size of colonies seen. Further classification of bacteria was conducted by streaking the isolated colonies onto different types of agar that help assist with classification based on the type of respiration that the bacteria undergo and the nutrients the strain of bacteria uses/consumes. The agar used in this experiment includes MacConkey Agar and Eosin-Methylene Blue Plates. In the presence of coliforms (suspected strain growing in agar), Gram-negative bacteria from the isolated colonies would grow on both plates, and depending on the color of the colonies and the number present, the strains can be narrowed down using reference charts.
IMVIC test: Furthermore, Gram-negative bacterial species can be narrowed down through the IMViC tests, a series of testing to identify a specific species of coliform. The test starts with an Indole Test, which involves the differential agent, Kovac’s Reagent. This allows the presence of indole to be detected from the catabolism of tryptophan (which is present in the solution) by the enzyme tryptophanase. The second and third tests are the Methyl Red and Voges-Prausker tests which are used to determine if the bacteria use a Mixed Acid Fermentation Pathway or the 2,3-butanediol fermentation pathway, respectively [22]. In the case of coliforms, the bacteria use only one of either fermentation pathway. Because of this, if the Methyl Red test is read at a later time, a case of a double negative or a double positive result may occur. From this, it cannot be determined that the bacterium is not a coliform. Lastly, Simmon’s Citrate Agar slant is used to determine whether the bacterial species are capable of metabolizing sodium citrate which serves as the sole source of carbon as the agar contains ammonium dihydrogen phosphate. The results of all these tests help narrow down the species of bacteria based on characteristics and respiration types of the bacterial sample [23]. If conclusions can be made based on the results from some tests, then further testing will not be performed [24].
Gram-Staining: To confirm the bacteria isolated are Gram-negative, we performed Gram staining on isolated colonies. This staining procedure allows one to clearly distinguish Gram-positive from Gram-negative bacteria by using a crystal violet-iodine complex stain and a safranin counterstain [25]. The cell walls of Gram-positive bacteria retain a purple coloring (based on the crystal violet stain), while Gram-negative bacteria decolorize after the addition of ethanol and appear pink in color (based on the safranin stain) [25]. This difference in color is clearly visible under a microscope.
RESULTS
From the MBRT tests, we were able to determine if the milk was contaminated with the presence of microorganisms. This was primarily done based on how long it took the tubes at 30°C to lose their blue coloring and turn white, signaling microbial activity. We observed the tubes for two hours and took observations/pictures at 30-minute intervals. This was done for all three milk samples prior to and after the expiry dates. There was a very slow and delayed color loss in non-expired samples, suggesting that the milk samples were not contaminated before expiring and were pasteurized, sealed, and sterilized properly (Fig. A).
We found some color loss in all expiring samples (on expiry date), although at different degrees. Organic Valley milk was lighter in color compared to the Producers milk sample, but it still had a blue hue. The Producers sample turned a light blue color with white chunks at the top and bottom of the tube. The Lucerne sample lost all of its blue coloring and became white in color, our first indicator of microbial presence and growth. This can be seen in Fig. B, which has the samples in order fromOrganic Valley, Producers, and Lucerne. Based on BIS 1479 grading, the MRBT tests showed that the quality of Lucerne milk was poor to fair due to the rate of color loss [26].
All three milk samples were inoculated onto nutrient agar plates 2 days before expiry, the day of expiry, and 2 days after expiry in order to observe any possible growth from microorganisms present. There was no bacterial growth on any of the agar plates before expiry and the day of expiry (Fig. C and Fig. D). This suggests that the microbes were not present or active on these days. When samples were inoculated after expiry, fungal growth, unlikely to be related to streaking, was seen on the Producers milk sample’s Nutrient Agar while bacterial colonies were visible on the Lucerne milk sample’s Nutrient Agar plate (Fig. E). Additionally, no bacterial colonies were isolated on Organic Valley milk samples.
Bacterial cultures did not grow until 5 days after the expiration date of the milk products, possibly due to pasteurization and processing before consumer sale (Fig. F).
Considering the bacterial growth, only the Lucerne milk sample was taken further for testing and confirming the presence of coliforms. To isolate and identify the bacteria, we performed the indole and citrate tests from the IMViC protocol (Table A, Fig. J, and Fig. K) first alongside inoculations in EMB agar (Fig. H) and MacConkey agar. (Fig. I). In order to inoculate the samples, we first let them grow in the Lauryl Tryptose Broth (Fig. G) so that we could isolate coliforms to be tested on through various experiments.
| Lauryl Tryptose | EMB Agar | MacConkey Agar | Indole Test | Citrate |
| + | + | – | – | + |
The Gram staining technique showed that the bacteria were gram-negative. This was determined by observing the color the cell walls of the bacteria adapted. Since the bacteria adapted a red color, we can tell the bacteria found in the Lucerne milk sample are gram-negative, therefore, confirming it is a coliform. (Fig. L)
DISCUSSION
Working with different samples of milk was highly effective because we were able to observe how different brands and types of milk acted differently for the growth of microorganisms and bacteria. We found that pasteurization of milk is highly effective. The pasteurization was able to prevent the growth of bacteria and common microorganisms in the milk. Because of this, the milk remained healthy and safe to consume at various intervals along the expiration dates. From the various bacterial and microbial tests performed, we found that the quality of the milk remained relatively consistent around the expiry date and even a few days after the expiration date of the milk samples.
Pasteurized milk samples showed no microbial growth at all before the expiration date, demonstrating that milk that is pasteurized generally tends to be safe for consumption, at least before the expiration date. Because no microbial growth was observed, we can conclude pasteurization of milk is one of the most effective ways to prevent the growth of microorganisms in milk.
Additionally, observation of the samples after the expiration date led to the conclusion that no microbial growth was present in the milk sample until five days after the date of expiration. This finding was surprising because the date of expiration generally is set to indicate a date after which the milk is no longer safe to consume. However, our results indicate that pasteurized milk was perfectly safe to consume until five days after expiration dates.This discrepancy is likely because the expiration date of milk is determined in large batches, and the date may not be representative of each carton or container of milk within the batch.
Microbial growth occurred in the pasteurized and expired milk samples five days after the expiration date. There were no bacterial colonies found in both the samples of Producer’s or Organic Valley’s expired and pasteurized milk, suggesting that five days post marked expiration, the milk would still be safe to ingest as the bacterial and microbial count of bacteria was significantly low and not present.
Unlike the expired samples of Organic Valley and Producer’s milk, which showed no bacterial growth, the expired sample of Lucerne milk showed a significant amount of bacterial colonies, indicating that the Lucerne milk would not be safe to ingest five days after expiry. However, because bacterial contamination was only found almost a week after expiration, it would be perfectly safe to consume Lucerne milk before the expiration date, or even up to a few days after expiry. The type of microorganisms present in the expired sample of Lucerne milk was not E. coli based on the biochemical assays performed. Further tests are necessary to determine which exact species and strain of bacteria it is.
The results we found were close to our hypothesis because it was hypothesized that pasteurization of milk significantly decreases the number of microorganisms present in that sample of milk. Two out of the three samples that were used showed no signs of microbial growth, even a week after expiration. Only one sample showed microbial growth, and that too only after five days.
Together, these findings demonstrate that pasteurization of milk is highly effective in most cases and that pasteurized milk generally leads to less microbial growth in the milk; however, the data also underscores the importance of microbial testing by health officials.
CONCLUSION
In this research, we used different tests to determine the quality of milk before and after the expiration dates set by the manufacturers. Samples from three different brands of milk were used to test their quality. We believed that the pasteurization method would be sufficient enough to prevent the milk samples from producing copious amounts of bacteria, fungi, etc before expiration. The expiration dates were thought to be mass-produced and hence not exactly accurate. We noticed that the Lucerne milk gained bacteria and microorganisms much quicker than any other sample. Milk before expiration generally tends to have none or fewer microorganisms compared to milk that has already expired. In our study, we confirmed this- as two of the three milk samples didn’t grow any bacteria at all until five days after the expiry date. After five days, significant amounts of bacterial growth were found. Pasteurization is an effective way of making milk safe to consume and the expiration dates- although not completely accurate down to exact days, it was close enough to that day to serve its purpose.
ACKNOWLEDGMENT
We would like to thank the Olive Children Foundation and the sponsors of the Aspiring Scholars Directed Research Program for organizing and providing resources throughout the program. We would also like to thank the faculty and staff of Fremont STEM for their assistance throughout the project. We would especially like to thank Ms. Hiral Dantara, Mr. Albert Chen, and Mr. Edward Njoo for their assistance and guidance in the completion of our research.
AUTHOR INFORMATION
Present Address Aspiring Scholars Directed Research Program, 43505 Mission Blvd, Fremont, CA 94539
Author Contributions All research members contributed equally to the research done and the writing of this paper.
Notes This work was conducted over a two-month period.
ABBREVIATIONS
- IMViC: Indole test, Methyl red test, Voges-Proskauer test, Citrate test
- MBRT: Methylene Blue Reductase Test
- EMB: Eosin Methylene Blue
- PPC: post-processing contamination
- MPN: Most Probable Number
- cfu/ml: colony-forming unit per milliliter
