Progress 10/01/21 to 09/30/22
Outputs
Target Audience:Scientific community and the dairy processing industry. Changes/Problems:None What opportunities for training and professional development has the project provided?This year I was able to attend the ASM Microbe meeting held in Washington DC and had several undergraduate students attend and share their research during the poster session. I also was an invited panelist for a roundtable on dairy spoilage at the ADSA annual meeting which I attended virtually. I also continued to teach sections of the short and advanced cheese making courses offered through the Western Dairy Center. How have the results been disseminated to communities of interest?Data from objective 2 and 4 were shared at the BUILD Dairy annual meeting held in Provo Utah and initial results for objective 3 were presented at the ASM Microbe meeting held in Washington DC and the ADSA annual meeting held in St. Louise. What do you plan to do during the next reporting period to accomplish the goals?I have received funding to complete objective 3 and have a master's level student conducting the research. Manuscripts are being prepared for objectives 3 and 4 and will be submitted to the Journal of Dairy Science this coming year.
Impacts
What was accomplished under these goals?
I currently have one project that has concluded to fulfill objective one which was published in the Journal of Dairy Science, objectives 2 and 4 have been complete and the students successfully defended their thesis this December. I have received funding for objective 3 and have a graduate student to complete the work on this objective. The progress that I have made on my project objectives are summarized below. 1. Understand the growth, survival, and death of lactococcal starter cultures in cheddar cheese during cheese production and storage using flow cytometry and classical agar plating techniques. This objective has been completed and the data that was collected from the research has been published in the Journal of Dairy Science. Briefly, two strains of L. lactis(SSM 7605, SSM 7436) and two strains of L. cremoris (SSM 7136, SSM 7661) were selected. A standardized Cheddar cheese make procedure was used that included a 38°C cook temperature and salting levels of 2.0, 2.4, 2.8, 3.2 and 3.6% from which were selected cheeses with salt-in-moisture (S/M) levels of 3.5, 4.5, and 5.5%. Vats of cheese were made using each strain on its own as biological duplicates on different days. Starter culture numbers were enumerated by plate counting during cheese making and after 6 d storage at 6°C. Flow cytometry with fluorescent staining by SYBR® Green and propidium iodide was used to determine the number of live and dead cells in cheese at the different salt levels. Differences in cheese make times were strain dependent rather than subspecies dependent. Even with correction for average culture chain length, cheeses made using L. lactis strains contained ~4 times (~ 0.5 log) more bacterial cells than those made using L. cremorisstrains. None of the four strains used in this study were influenced by the amount of salt added to the curd. The higher pH of cheeses with higher salting levels was attributed to those cheeses having a lower moisture content. Based on flow cytometry, ~5% of the total starter culture cells in the cheese were dead after 6 d of storage. Another 5 to 15% of the cells were designated as being live, but semi-permeable, with L. cremorisstrains having the higher number of semi-permeable cells. 2. Establish the metabolic capabilities of lactococcal starter cultures in cheddar cheese using next generation sequencing techniques. Several lactococcal strains were isolated from cheddar cheese curd and whole genome sequencing was conducted. These genomes were analyzed to identify genes for proteolytic enzymes. Based on this data, we are proposing a new proteolytic class of lactococcal strains. These strains were then used to develop a method based to differentiate L. lactis and L. cremoris using rtPCR with amplification of a set of genes in the ADI pathway. The L. cremoris strains lack this pathway and the L. lactis strains have genes for the pathway. Small batches of Cheddar cheese were produced in the USU Dairy Products Laboratory using single strain starters of each species and a 50:50 blend. Samples were taken during the cheese make and at intervals for 12 weeks after production. The samples were then processed for total DNA isolation and the DNA was used to quantify the level of each culture. Traditionally, Cheddar cheese has been produced using L. cremoris strains because the cheese is less likely to develop bitterness during the aging process. To be more productive, most modern Cheddar producers use a blend of L. lactis and L. cremoris or just L. lactis during production. The L. lactis strains are more resistant to high sodium levels and will continue to produce acid after the cheese curd has been salted. The allows producers to run more cheese vats during a production day, but increases the likelihood of the cheese developing bitter flavors during aging. The data showed that this method we used is valid and has the potential to track the different species during cheese production and storage. This will allow us to understand the how these cultures contribute to the development of bitterness in aged cheese. This data resulted in a master's thesis which was successfully defended, and the thesis is being developed into a manuscript for publication in the Journal of Dairy Science. 3. Determine the metabolic capabilities of the most common non-starter lactic acid bacteriaLatilactobacillus curvatusin cheddar cheese. Initial experimental design has been done for this objective, and funding has been secured through the BUILD Dairy program to support a master's student. The Student started fall 2022 and completed the initial fermentations which show that Lat. curvatus has the ability to make reuterin under anaerobic conditions with the addition of glycerol. The student is currently preparing to conduct a challenge test to see if the fermentate is inhibitory to a panel or yeast and molds that have been isolated from dairy products. 4. Test the efficacy of potential adjunct cultures to decrease gas splitting and cracking quality defects in cheddar cheese at the pilot plant scale. This objective is complete, and the graduate student has successfully completed their thesis defense. The results are summarized below. Heterofermentative non-starter lactic acid bacteria (NSLAB) can pose a major problem in the dairy industry by causing late-stage gas formation defects in Cheddar cheese, which are characterized by slits, cracks, and blown bags. Slits and cracks make the cheese more difficult to shred and slice, and, along with the blown bags, cause the cheese to be less appealing to the customer. These defects can also cause the cheese to be downgraded to a lower margin product, which reduces manufacture profits. Heterofermentative NSLAB have the ability use six-carbon sugars, like galactose, to produce carbon dioxide. Recently, starter cultures like Streptococcus thermophilus are used to increase the rate of acid production during Cheddar cheese production, which increases the risk of gas production due to its inability to ferment galactose. The primary objective of this research was to use previously identified galactose positive and lactose negative protective adjunct cultures, as well as other protective adjunct cultures that we believed decrease the amount of gas produced by heterofermentative NSLAB. The adjunct cultures effect on gas production was determined by challenging them with Levilactobacillus brevis, Limosilactobacillus fermentum, Lentilactobacillus parabuchneri,and Paucilactobacillus wasatchensis.These cultures are all known gas producing heterofermentative NSLAB.The selected adjunct cultures were Lactocaseibacillus rhamnosus, Lactocaseibacillus paracasei, Pediococcus acidilactici,and Latilactobacillus curvatus.We added these adjunct cultures to the milk at the beginning of the cheese make along with the S. thermophilusstarter culture. The following day the cheese was ground and inoculated with individually grown tubes of our heterofermentative NSLAB in duplicate at 104 CFU/g totaling 10 samples including controls. These 10 samples were then pressed back into blocks with each sample cut into 7 sub-samples of 450 g each, and vacuum sealed. Gas levels were checked weekly for 16 weeks for all samples. This process was done in duplicate for every protective adjunct culture. Results showed there is potential for using protective adjunct cultures for reducing late gas production in Cheddar cheese. Most notably, there were reductions in gas production when Lat. curvatus was challenged with Lev. brevis,and when Lcb. rhamnosusand Lcb. paracasei were challenged with Pa. wasatchensis. There was also an increase in gas production and the gas production rate when P. acidilacticiwas challenged with Pa. wasatchensis.
Publications
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Progress 10/01/20 to 09/30/21
Outputs
Target Audience:Scientific community and the dairy processing industry. Changes/Problems:None What opportunities for training and professional development has the project provided?This year I was able to help teach sections of the short and long cheesemaking coursed offered through the Western Dairy Center, and also section on food safety for a small dairy producers conference supported by Dairy West. How have the results been disseminated to communities of interest?The research that was conducted for objective one has been complete and the manuscript has been accepted for publication in the Journal of Dairy Science. One of the graduate students who is working on objective 4 was able to present his research at the American Dairy Science Association annual meeting which was held virtually during 2021. What do you plan to do during the next reporting period to accomplish the goals?The plan for the next reporting year is to continue to pursue funding opportunities to advance the research on my listed objectives. The data collected for objective 4 has been completed and results from this objective are being used to prepare a manuscript for publication in a peer reviewed journal. The cultures that will be used for cheese making for objective 2 have been selected and vats of cheddar cheese will be produced spring 2022.
Impacts
What was accomplished under these goals?
During this report period I was able to make progress on my UAES research project. I currently have one project that has concluded to fulfill objective one, and two graduate students who are completing their research to fulfill objectives 2 and 4. I am currently seeking funding for objective 3. The progress that I have made on my project objectives are summarized below. 1. Understand the growth, survival, and death of lactococcal starter cultures in cheddar cheese during cheese production and storage using flow cytometry and classical agar plating techniques. This objective has been completed and the data that was collected from the research has been published in the Journal of Dairy Science. Briefly, two strains of L. lactis(SSM 7605, SSM 7436) and two strains of L. cremoris (SSM 7136, SSM 7661) were selected. A standardized Cheddar cheese make procedure was used that included a 38°C cook temperature and salting levels of 2.0, 2.4, 2.8, 3.2 and 3.6% from which were selected cheeses with salt-in-moisture (S/M) levels of 3.5, 4.5, and 5.5%. Vats of cheese were made using each strain on its own as biological duplicates on different days. Starter culture numbers were enumerated by plate counting during cheese making and after 6 d storage at 6°C. Flow cytometry with fluorescent staining by SYBR® Green and propidium iodide was used to determine the number of live and dead cells in cheese at the different salt levels. Differences in cheese make times were strain dependent rather than subspecies dependent. Even with correction for average culture chain length, cheeses made using L. lactis strains contained ~4 times (~ 0.5 log) more bacterial cells than those made using L. cremorisstrains. None of the four strains used in this study were influenced by the amount of salt added to the curd. The higher pH of cheeses with higher salting levels was attributed to those cheeses having a lower moisture content. Based on flow cytometry, ~5% of the total starter culture cells in the cheese were dead after 6 d of storage. Another 5 to 15% of the cells were designated as being live, but semi-permeable, with L. cremorisstrains having the higher number of semi-permeable cells. 2. Establish the metabolic capabilities of lactococcal starter cultures in cheddar cheese using next generation sequencing techniques. Cultures have been selected for the cheesemaking, and initial data has been collected on the use of selected primers for RT-PCR for quantifying the amount of starter bacteria in the samples. 3. Determine the metabolic capabilities of the most common non-starter lactic acid bacteria Latilactobacillus curvatus in cheddar cheese. Initial experimental design has been done for this objective, and a grant has been submitted to the BUILD Dairy program for funding. 4. Test the efficacy of potential adjunct cultures to decrease gas splitting and cracking quality defects in cheddar cheese at the pilot plant scale. All of the research work for this objective has been completed and the data is currently being analyzed for statistical significance. A manuscript is in preparation and should be submitted for publication in 2022.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2021
Citation:
Presentations
Crompton, R. (Presenter & Author), Oberg, T. S. (Author Only), ADSA Annual meeting, "THE USE OF PROTECTIVE LACTIC ACID BACTERIA ADJUNCT CULTURES TO DECREASE THE INCIDENCE OF GAS DEFECTS IN CHEDDAR CHEESE PRODUCTION," American Dairy Science Association, Online. (July 2021)
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Progress 06/19/20 to 09/30/20
Outputs
Target Audience:Scientific community and the dairy processing industry. Changes/Problems:Currently there are no problems or proposed changes to my current UAES project. What opportunities for training and professional development has the project provided?There have been several training and development opportunities during this reporting period listed below: - Students trained in cheddar cheese production and dairy equipment sanitation. - Student trained in bioinformatic and big data analysis and management. - Students trained in microbial media production and food microbiology enumeration methods. How have the results been disseminated to communities of interest?All of the professional meetings that we planned to attend and present research at were cancelled due to the Covid-19 global pandemic. What do you plan to do during the next reporting period to accomplish the goals?The plan for the next reporting year is to continue to pursue funding opportunities to advance the research on my listed objectives. The data collected for objective 4 will be completed this upcoming spring and data analysis will be complete during the summer, and results from this objective will be used to prepare a manuscript for publication in a peer reviewed journal. The cultures that will be used for cheese making for objective 2 will be selected and vats of cheddar cheese will be produced. Hopefully there will be some professional meetings that the current research will be presented at.
Impacts
What was accomplished under these goals?
During this report period I was able to make progress on my UAES research project. I was able to secure two grants through the BUILD Dairy program totaling $217,300. These grants include funding to support two masters level students which started working in my research lab in July 2020. Both students are currently developing methods and collecting data for their research projects. Listed below are my project objectives with the progress that was made during this report period: 1. Understand the growth, survival, and death of lactococcal starter cultures in cheddar cheese during cheese production and storage using flow cytometry and classical agar plating techniques. - No progress was made on this objective. 2. Establish the metabolic capabilities of lactococcal starter cultures in cheddar cheese using next generation sequencing techniques. - Wild type cultures of both L. lactis and L. cremoris were isolated from commercially produced cheddar cheese curd. - The whole genome sequence of these cultures was obtained through the isolation, sequencing and assembly of the sequence. The sequence was generated at the USU Center for Bioinformatics. The assembled genomes were then annotated using the open source annotation software RAST, and the species of each organism was confirmed by BLASTing the 16S ribosomal gene DNA sequence against the NCBI sequence database. 3. Determine the metabolic capabilities of the most common non-starter lactic acid bacteria Latilactobacillus curvatus in cheddar cheese. -No progress was made on this objective. 4. Test the efficacy of potential adjunct cultures to decrease gas splitting and cracking quality defects in cheddar cheese at the pilot plant scale. - Four adjunct cultures were selected for use as protective cultures. - 10 vats of cheddar cheese were produced at the Gary H. Richardson dairy products laboratory at USU. Each vat produced 6 twenty-pound blocks of cheese. - One block of each vat was sequestered and is currently aging at 8oC. - The remaining cheese blocks were ground and each inoculated with a different HFLAB which have been shown to cause gassing defects in commercial cheddar cheese. - Samples were vacuumed packed and aged at 14oC for 16 weeks. - These cheese samples were enumerated for starter and non-starter LAB at 1 and 8 weeks, and the amount of gas produced was measured weekly.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2020
Citation:
Presentations
Leatham, D. (Presenter & Author), Oberg, T. (Author Only), Oberg, C. J. (Author Only), ASM Intermountain Branch Meeting, "Antimicrobial Metabolites of Lactobacillus curvatus," American Society for Microbiology, Odgen, Utah. (December 5, 2020)
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