Dairy Science – Alcaine Research Group

April 21, 2021kgj5

Taste a Whey Better Beverage

RESEARCH SPOTLIGHT: Characterization of Sensory Profiles of Yogurt Acid Whey Fermented with Five Different Yeasts

This week, we are highlighting the work of MPS student, Rossie Luo. If you have been following along with the research spotlights, you may have noticed that ARG does a lot of research investigating novel fermentations of dairy effluents to valorize waste products and create a more sustainable process. But how do these fermented beverages actually taste?? Rossie’s research sets out to answer that question!

Rossie’s work is centered around evaluating the sensory profile of fermented beverages made from yogurt acid whey. Unlike sweet whey that can be used to make whey protein powder, yogurt acid whey typically has limited value-added applications and is mainly used as animal feed or as a fertilizer. Yet, acid whey still has residual lactose, making it a suitable substrate for yeast fermentation. The goal of her research is to ferment yogurt acid whey with five different yeasts to evaluate the sensory profiles that each yeast creates during fermentation.

To do this, Rossie set up fermentations with five different yeast strains and monitored the density, pH, and cell concentration throughout the duration of the fermentation to characterize the fermentation profiles of each yeast. She also compared the sugar, ethanol, and organic acid concentration at the beginning and end of fermentation for each yeast. What she saw is that each yeast creates a unique fermentation profile for the yogurt acid whey!

Once the fermentations were complete, she set out to characterize the sensory profiles of each beverage prototype. Through the Sensory Evaluation Center at Cornell, a focus group was conducted and was followed by an individual questionnaire. During the focus group, participants were guided to discuss various aspects of their sensory perception of the beverage prototypes such as aroma, flavor, mouthfeel, and aftertaste. A list of sensory descriptors was then generated and used as metrics for the individual questionnaire to evaluate the prototypes. It turns out that these yeasts were able to impart different and unique flavors to the fermented yogurt acid whey!

This study provides important insights that can guide further product development of a fermented acid whey beverage. It is exciting to think that we can be part of a more sustainable future with delicious and nutritious fermented yogurt acid whey beverages!

^{Sensory profiles of the fermented acid whey beverages [1]}

[1] Siyi (Rossie) L, Timothy A. D, Dana D, Samuel D. A. (2021) Characterization of the Fermentation and Sensory Profiles of Novel Yeast-Fermented Acid Whey Beverages. (manuscript in preparation).

April 14, 2021kgj5

Roses are Red, the Mozzarella is Blue…

RESEARCH SPOTLIGHT: Topical application of lactose oxidase to inhibit spoilage microbes that cause blue discoloration of mozzarella cheese

_{^{Blue discoloration of mozzarella cheese. Image retrieved from: https://foodinitaly.wordpress.com/2010/10/08/news-blue-mozzarella/}}

This week, we are highlighting the work of MPS student, Pablo Torres. He is investigating enzymatic-preservation techniques to stop mozzarella cheese from turning blue! In 2010, over 70,000 mozzarella balls in Italy turned blue as a result of bacterial contamination that created a blue discoloration on the fresh cheese ^[1]. It was quite a spectacle!

Microbial contamination is one of the largest factors adversely affecting the profitability of the dairy industry, as it shortens the shelf life and increases waste. Mozzarella is produced via the pasta-filata, or stretched curd, technique that is done in high temperature water. The curd is mechanically stretched to obtain its unique textural and melting characteristics. Once the mozzarella is formed, it is susceptible to bacteria that can grow at refrigeration temperatures, or psychrotrophic bacteria. One of the main psychrotrophic organisms of concern in the dairy industry is Pseudomonas spp. which can cause spoilage defects. Among these defects are the blue discoloration produced by Pseudomonas fluorescens in dairy products like fresh mozzarella or queso fresco. This blue discoloration has also been observed in non-dairy products like salmon or rabbit meat!

So, what are some ways that we can stop the cheese from getting this blue defect? Enzyme-based preservation technologies have been a novel method for preservation in the dairy industry. Enzymatic preservation can be a clean label approach to reduce spoilage and extend shelf life. An enzyme of interest is lactose oxidase, a naturally derived enzyme that hydrolyzes milk sugar into lactobionic acid and reduces oxygen to hydrogen peroxide. This can potentially work as an antimicrobial against Pseudomonas fluorescens and stop the formation of the blue pigment.

To analyze if lactose oxidase can reduce the blue discoloration, Pablo topically applies lactose oxidase to cheese that has been contaminated with Pseudomonas fluorescens. Throughout the duration of the experiment, he takes photos of the mozzarella and compares the treatments to a control sample.

A software is used to quantify color using the CIE, or L*a*b* color space. Colors are measured on a scale for values of L*, a*, and b* which are determined by the color wheel of human vision. L* represents the lightness of a sample, 0 representing black and 100 pure white, a* quantifies the green-red scale, and the b* axis representing blue-yellow values. With the data provided by the software, a comparison between the treatment samples and the control can be made to determine if the lactose oxidase is effective at reducing the bacteria that cause the blue discoloration.

So far, lactose oxidase has been an effective enzymatic-preservation technique, resulting in reduced blue color in treated samples!

[1] Greenhalgh, M. (2010). Bacterial Contamination Caused Blue Mozzarella. Retrieved from: https://www.foodsafetynews.com/2010/06/bacterial-contamination-caused-blue-mozzarella/

April 7, 2021kgj5

Drink It Our Whey!

RESEARCH SPOTLIGHT: Optimizing alcoholic fermentations to develop new products from whey permeate

This week, we are highlighting the work of PhD student, Viviana Rivera Flores. Viviana is working on optimizing the fermentation process of whey permeate under anaerobic conditions to develop low-alcohol beverages with potential functionality. Whey permeate is the dairy effluent that results after obtaining protein concentrates from cheese whey. For every pound of cheese produced, about seven and a half pounds of permeate are created.

While whey permeate has gained attention in the food industry as an additive, novel biomanufacturing technologies can open avenues for greater valorization of this product. In oxygen-deprived atmospheres, yeast can convert lactose from this substrate into ethanol and galactose, offering a sustainable and flexible alternative. Galactose acts as a prebiotic precursor and provides energy without altering our sugar balance, hence a beverage with this sugar would have functional properties. On the whole, anaerobic fermentation of whey permeate is a versatile process from which we can generate bioethanol, recover galactose, or develop novel beverages. Three possible products from only one raw material!

General steps of her methodology include substrate preparation, yeast propagation, fermentation experiments, product analysis, optimization model development, and further process optimization.

Currently, Viviana is working on the optimization model of the fermentation process. She is evaluating the impact of different parameters (like temperature and time) on ethanol and galactose concentration. To make this possible, she works with a software that builds statistical models that take multiple factors to optimize the desired products. For better optimization, this software uses a design called response surface methodology, which is an efficient way to assess the individual impact of each fermentation parameter and understand their interactions. If ethanol and galactose are maximized, more efficient valorization and better functionality of this waste stream can be achieved.

To accomplish this, she has been running benchtop scale fermentations with multiple combinations of fermentation factors. This has led to over 160 individual fermentations!

So far, her results demonstrate that it is possible to adjust multiple parameters to maximize ethanol and galactose production from whey permeate. Once she finalizes the process optimization stage, her next steps will be to explore different formulations to develop a novel beverage and investigate this process at larger scales.

So, if you are a health-conscious consumer looking for a better-for-you, refreshing hard seltzer that’s also committed to a more sustainable environment, stay with us!

March 31, 2021kgj5

In Queso Emergency

RESEARCH SPOTLIGHT: Using Lactose Oxidase as a natural antimicrobial to inhibit growth of Listeria monocytogenes in Queso Fresco

This research spotlight highlights the work of master’s student Brenna Flynn. Brenna is investigating whether a naturally derived enzyme, lactose oxidase, can inhibit the growth of Listeria monocytogenes in queso fresco. Listeriosis is a high-risk infection caused by the consumption of L. monocytogenes which predominantly affects those with compromised immune systems, pregnant women, and the elderly. It also has a high lethality rate which makes it a main cause for concern in food safety.

^{Listeria monocytogenes colonies on MOX plates}

L. monocytogenes is a problem for the dairy industry because it commonly contaminates products after pasteurization. Many dairy products are ready-to-eat (RTE), meaning that there’s no further kill step after pasteurization to reduce Listeria counts before the consumer eats the product! In addition, queso fresco has a near-neutral pH with a high water content, making it an ideal environment for pathogens to thrive if the cheese becomes contaminated. In ARG, we are trying to find an effective control method to reduce or eliminate L. monocytogenes in queso fresco to mitigate future L. monocytogenes outbreaks and keep consumers safe!

^{Preparation of lab-scale queso fresco}

With increased consumer demand for clean-label ingredients, enzymatic antimicrobials can be an option for producers. Lactose oxidase is a naturally derived enzyme that oxidizes the sugar in milk, lactose, into lactobionic acid, while reducing oxygen into hydrogen peroxide. This reaction is an activator of a native antimicrobial system in milk. Hydrogen peroxide itself is also a widely used antimicrobial that is generally recognized as safe at low concentrations in food. In previous studies, hydrogen peroxide has shown to be an effective inhibitor of L. monocytogenes in queso fresco.

Brenna has been making lab-scale queso fresco to test if lactose oxidase has antimicrobial properties. She has run multiple experiments, using lactose oxidase as both an additive to the milk during the cheesemaking process and as a topical application. She then inoculates the cheese with L. monocytogenes to determine if lactose oxidase has an antimicrobial effect.

So far, her experiments have shown that lactose oxidase is effective at inhibiting the growth of L. monocytogenes! Lactose oxidase could potentially be used as an antimicrobial in the dairy industry to keep consumers everywhere safe!

March 24, 2021kgj5

No Need to Cry Over Spoiled Milk!

RESEARCH SPOTLIGHT: IDing of food spoilage organisms for dairy manufacturers

This research spotlight highlights the work that Lab Technician Tim DeMarsh is doing to help prevent spoilage microbes from causing issues in the dairy industry. Contamination with spoilage microbes can result in food waste and loss of revenue for dairy producers. Remediating spoilage issues can be difficult, especially when producers don’t know which microbe is responsible. It’s much easier to create solutions when manufacturers can specifically target the organism that is causing the problem. Producers can send their contaminated food samples to the Alcaine Research Group where Tim identifies the offending species. Using this information, the manufacturer can develop strategies to mitigate this specific species and prevent future instances of spoilage.

To identify the organism, it must first be isolated from the food sample. The microbe is grown up on a petri dish for isolation and then its cells are split open, exposing its DNA for extraction. Tim then uses a technique called Polymerase Chain Reaction (a.k.a. PCR; the same process used for COVID-19 tests!) to make billions of copies of the organism’s DNA. The PCR product is loaded into an agarose gel, a scientific version of your favorite Jell-O dessert. Then, the gel is exposed to an electrical field. Since DNA is a negatively charged molecule, it migrates through the gel to the positively charged side, creating bands of DNA.

To make these bands really stand out, the gel is stained with ethidium bromide, a chemical that binds to DNA and fluoresces under UV light. When the gel is exposed to UV light, colorful bands of DNA aggregates are visible, verifying that Tim’s PCR process was successful. He now has billions of copies of the pesky spoilage microbe’s DNA! The DNA is purified and sent to another lab at Cornell that sequences the DNA for him, creating a genetic fingerprint for the organism.

When Tim receives the DNA sequence data, it must be cleaned up a little, and then he compares the sequence to an NCBI database that contains over 1.3 billion unique DNA sequences. The database identifies close matches to the DNA sequence and provides names of organisms that are similar. If the fidelity of the match is 99% or higher, he can be relatively confident that his organism matches the organism in the database, and he has found the name of the species causing the spoilage issues!

The name of the microbe is then given to the dairy processor, and they can create a targeted approach to remediating the issue. This service provided by the Alcaine Research Group helps manufacturers ensure that they are producing high-quality, spoilage free products that consumers everywhere will enjoy!

March 17, 2021kgj5

Got Milk?

RESEARCH SPOTLIGHT: Fermenting surplus skim milk into a value-added product

This week, we are highlighting the work of master’s student Lucas Wise. Lucas is focusing on repurposing surplus skim milk into a value-added product. High prevalence of lactose intolerance in the world and increased demand for plant-based milk alternatives has caused a decline in skim milk consumption. For producers, this is resulting in financial loss and environmental damage from increasing skim milk waste. The goal of his project is to develop technologies to derive added value compounds, ingredients, and products from skim milk via the fermentation of lactose.

While traditional brewer’s yeast, Saccharomyces cerevisiae, can’t ferment lactose, alternative yeasts can externally break down lactose into its two smaller subunits, glucose and galactose. Non-Saccharomyces yeasts can also selectively ferment the glucose, leaving galactose behind to be recovered!

*Lactose cleavage into its two monomers, glucose and galactose*

Galactose is a low glycemic index sugar, meaning that it doesn’t cause a huge spike in blood sugar levels after its ingestion. Galactose can be used to make healthier snacks and beverages, serve as a pre-cursor to other “rare” sweeteners like tagatose, and can be used in pharmaceuticals and health-promoting galactooligosaccharrides, which are prebiotics.

The anaerobic fermentation of lactose also produces ethanol, the ingredient in all of our favorite drinks!

The fermentation process can leverage the lactose in discarded skim milk and provide an alternative method of upcycling this dairy waste, easing the burden of waste on dairy producers. Companies could avoid major financial losses and reduce their environmental impact because the skim milk surplus gets repurposed.

Currently, Lucas is in the screening stage, using a variety of yeast species to determine the best process performance parameters and improve the fermentation stability and sensory properties of the product. This will be followed by an optimization seeking to maximize ethanol and galactose yields from the fermentation. This work could potentially be used to create a stand-alone fermented beverage!

March 10, 2021kgj5

Holey Cow!

RESEARCH SPOTLIGHT: Late-blowing defect in cheese caused by Clostridium tyrobutyricum

This week, we will be highlighting a project that PhD Student Margarita Valdiviezo is working on with assistance from one of our lab techs, Dana. They are working with an interesting organism that has long acted as a pest to cheese producers- Clostridium tyrobutyricum. C. tyrobutyricum is a spore forming bacteria; this means that it is able to survive pasteurization temperatures that kill off most other unwanted microbes. Once conditions are favorable, and vegetative cells begin to grow in the cheese from the spores. Butyric acid and gas are produced as a byproduct of their metabolism. This causes a defect knowing as “late-blowing”. As you can see in the photo, this causes cracks and bubbles to form in the cheese that are not desirable.

Samples were received from a cheese producer to isolate and confirm the identity of C. tyrobutyricum, as well as to develop a sporulation protocol that Margarita will be able to apply to her research. Margarita plans to develop biotechnological strategies that will reduce spore forming bacteria in organic dairy products.

Margarita and Dana are still in the process of attempting sporulation. The process has been as follows: a sample of the cheese was digested in a stomacher and plated. Then, an overnight tube was made with an isolated colony from the plate. The following day, 1mL of that tube was transferred to a biphasic media bottle and allowed to incubate. All of this took place in an anaerobic chamber, as C. tyrobutyricum is a very oxygen sensitive organism and can only grow in anaerobic environments. The next step in this process is to confirm the presence of spores using a phase-contrast microscope.

March 2, 2021kgj5

What’s Shakin’?

RESEARCH SPOTLIGHT: Fermentation of Dairy Waste Stream into Value-Added Product

For our first research spotlight, we are highlighting some of the novel dairy fermentation work going on in ARG. Marie Lawton and Kate Jencarelli have been aerobically fermenting a synthetic media supplemented with lactose to emulate dairy effluents. Acid whey, whey permeate, and milk permeate are by-products of dairy processing. They’re generally low in solids but have high levels of the milk sugar lactose remaining.

These by-products are typically waste and must be treated and disposed or they are used as animal feed. But what if we were able to convert this dairy waste into something great?

How can we upcycle dairy waste and create a value-added product?

Using the tools of microbiology, we can convert the milk sugars into something of value and help dairy processors turn their waste into a functional product.

With the rise in consumer trends for functional beverages, products like kombucha with live cultures have increased in popularity. Why couldn’t we make a kombucha like product out of these dairy effluents?

Acetic acid is the compound that gives kombucha its signature flavor, and dairy effluents are also packed with vitamins and minerals. Fermentation of these dairy effluents could break into a new niche in the market, and help dairy producers everywhere recycle their waste into a new profitable product!

Anaerobic fermentation of these dairy effluents is also being investigated in our lab. This process produces ethanol and could be another means of upcycling dairy waste. Research is ongoing to determine how to optimize acetic acid and ethanol production from the dairy waste stream.

March 2, 2021kgj5

Fermenting Ideas for Dairy Culture

We are a lab in the Department of Food Science at Cornell University in Ithaca, New York. We have a passion for the the world of dairy science and fermentations. Whether it’s chasing phage tails, teaching yogurt, or fermenting something wild…we are all about trying something new.

We hope you enjoy following along with the research and adventures we undertake. Please reach out if you have any questions, ideas, or just want to say hello!

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