Progress 10/01/19 to 09/30/20
Outputs
Target Audience:The target audience for this project included dairy processors, dairy equipment manufacturing companies, and dairy separation membrane users. The research outcomes were also shared with the industry personnel and academia, in addition to inclusion in the teaching and instructional content for undergraduate and graduate teaching in the Dairy and Food Science Department. Changes/Problems:Lab work on this project was completely stopped during March to May 2020. We were able to restart partial lab work on June 1. The time while working remotely was utilized for data compilation, analysis, completion of reports, and writing scientific manuscripts. What opportunities for training and professional development has the project provided?The project provided opportunities to train 1 Post Doc, 3 Ph.D., 3 MS, and 2 undergraduate students on advanced microbiological and dairy processing techniques. All these students were mentored on day-to-day basis by providing them opportunities to work with the faculty advisor. The Post Doc and graduate students were also provided professional development opportunities by supporting their travel and participation in regional and national conferences and professional meetings. Dr. Neha Singh. Post Doc. 2019-20. WGS approach for understanding environmental Listeria monocytogenes. Steve Beckman. Ph.D. Likely completion, Spring 2021. Quality of dairy ingredients as influenced by membrane processes. Shayanti Minj. Ph.D. Likely completion, Fall 2021. Development of dairy health foods using whey proteins and encapsulated probiotics. Taghreed Almalki. Ph.D. Likely completion, Spring 2022. Assessment of microbial biofilm formation of stainless-steel contact surfaces and weldments. Pratishtha Verma. MS. 2017-20. A natural antimicrobial from Bacillus subtilis as a bio sanitizer for resilient membrane biofilms. Ratul Kalita. MS. Likely completion, Fall 2021. Evaluating and modeling of sampling variability and sampling devices. Sheetal Jha. MS. Likely completion, Spring 2022. Development of a natural bio sanitizer for dairy plants. Allison Haltli, (BS Biology), 2020. 'Comparing Listeria species to form biofilms on different dairy floor types', undergraduate research project to complete the requirements of Biology major. Taylor Gilmore, 2020, (BS Dairy Production) 'Developing a novel whipped cream formulation containing WPH hydrolysate-encapsulated probiotics', undergraduate research project to complete the requirements of Dairy science. How have the results been disseminated to communities of interest?The results have been disseminated to the dairy industry and dairy farmers through Midwest Dairy Center meetings, the American Dairy Science Association national meeting, and the National Dairy Council and US Dairy Export Council national spore symposium. In all, three peer reviewed research articles, nine conference and workshop presentations, and two invited talks were delivered. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To improve microbial quality and shelf life of milk and dairy products Task 1.1: Development of a Natural Biosanitizer for More Effective Removal of Resilient Biofilms on Membranes. Task 1.2: To study biofilm formation on polished and unpolished stainless-steel contact surfaces in dairy industry. Task 1.3: Statistical evaluation of the robustness of microbiological sampling and cross-contamination potential of product sampling devices used in dairy Industry. Objective 2. To improve microbial safety of dairy processes Task 2.1: To control persistent Listeria in dairy processing environment by evaluating resident strains for disinfectant tolerance. Objective 3. To develop novel dairy products containing probiotics. Task 3.1: To develop a spray-dried health formulation based on whey protein hydrolysate and probiotics encapsulation.
Impacts
What was accomplished under these goals?
Objective 1: (90% Accomplished) Some microbes that grow within the biofilm that develops on dairy processing membranes acquire greater resistance to chemical cleaning products following prolonged use. This phenomenon was studied in the current investigation. An 18-month-old reverse osmosis membrane was procured from a whey processing plant. Membrane pieces (1 x 1 inch) were neutralized by dipping in Letheen broth and were then added to Tryptic Soy Broth (TSB). After mixing, samples were plated on Tryptic Soy Agar (TSA) to recover the constitutive microflora. Distinct colonies were identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, and Bacillus subtilis. These microbes were then co-cultured in pairs (15 combinations) to assess the emergence of predominance via competitive exclusion. Pure cultures of the respective isolates were spiked in the ratio of 1:1 in TSB, and incubated at 37? for 24 h, followed by plating on TSA. Bacillus subtilis emerged as predominant, with a mean log count of 7.22±0.22 CFU/ mL, which was also validated using the process of natural selection in a multi-species growth environment. Overall, five sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates at the end of each cycle. The fifth subsequent transfer detected the presence of only B. subtilis, thereby validating its predominance under the conditions of the experiment. Current cleaning and sanitation protocols may not be adequate for separation membranes, resulting in formation of resilient multispecies biofilms. The mature biofilms may result in a bacterial predominance with resilient strains. In our previous study, competitive exclusion revealed the predominance of B. subtilis within the membrane biofilm microflora. This study investigated the antimicrobial activity of the B. subtilis isolate as a potential cause of its predominance. Three culture isolates of B. subtilis were propagated in TSB at 37°C, and microfiltered at various time points to prepare cell-free extracts (CFE). The antimicrobial activities of CFEs were tested using an agar-well assay against the biofilm constitutive microflora. The experiments were conducted in triplicate, and means were compared for significant differences using a general linear mixed model procedure. The results indicated the highest antimicrobial activity of B. subtilis against other constitutive microflora developed at 12h incubation, with average inhibition zone sizes of 16.5-20.6 mm. Upon treatment with proteinase K, the CFE completely lost its antimicrobial activity, establishing the antimicrobial agent to be proteinaceous. Amino acid profiling revealed the total crude protein in CFE to be 51% (wt/wt), with the major constituent being glutamic acid (11.30% wt/wt). Freeze-dried CFE was thermally stable on exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min) and over a pH range of 3.0 to 6.3. Objective 2: (75% Accomplished) Three Listeria isolates from dairy plants - L. monocytogenes (Lm), L innocua (Li), and L. welshimeri (Lw) - were compared for their cell surface properties and adhesion on different floor types: clay brick, poured concrete, and grout. To evaluate cell attachment, different floor chips (1x1cm) were immersed in sterile distilled water, containing 6 logs/mL of the individual Listeria strain, and held for 1h at 22.4°C in a shaker incubator and plated on brain heart infusion agar (BHI). Cell surface hydrophobicity was determined by the hexadecane method. Zeta potential was determined by the Zeta sizer. Contact angle on the dairy floor types was measured using Sessile Drop device. Trials were conducted in triplicate, and means were compared by analysis of variance. The attachment study indicated the highest adhesion (log CFU/cm2) of all three isolates on poured concrete (Lm 3.54±0.04, Li 3.27±0.16 and Lw 3.70±0.24), as compared to grout (Lm 3.28±0.51, Li 2.67±0.41 and Lw 2.71±0.53), and clay brick (Lm 2.91±0.16, Li 2.73±0.04 and Lw 2.89±0.40). The water contact angles on all three surfaces were less than 90° (clay brick 43.3± 0.78, poured concrete 47.9±3.23 and grout 64.5±2.85) indicating they are hydrophilic and thus support microbial attachment. Of the three isolates, Lm and Lw resulted in a greater attachment than Li, which was also supported by their respective hydrophobicity values (20.13, 21.15, and 13.88%). Zeta potential values, however, were similar (-15.8, -16.2, and -15.5 mv, respectively) and did not appear to influence attachment. Listeria monocytogenes is a high-risk pathogen that can colonize and persist in dairy processing plants. The current study investigates the genetic determinants that could be associated with the biofilm forming ability of some environmental Listeria isolates. Three isolates from dairy processing environment, L. monocytogenes, L innocua, and L. welshimeri were compared for biofilm forming ability on dairy floors. Small sterile chips (1x1cm) of dairy bricks were submerged in sterile dairy effluents inoculated with respective Listeria sp., and held at 22.5°C for 48 h to form biofilms. Swab samples of chips drawn at the end of incubation were plated on BHI agar to enumerate biofilm-embedded cells. Means were compared using analysis of variance. Our results indicate a variability in the biofilm forming ability of the three isolates: L. monocytogenes (log cfu 2.50 ± 0.14/cm2), L. innocua (log cfu 2.72 ± 0.26 /cm2), and L. welshimeri (log cfu 3.32± 0.18/cm2). The three Listeria species were characterized using genetic determinants influencing colonization and biofilm formation. Genomes were assembled using CLC Genomics Workbench and resulting assemblies were used to identify MSLT types based on the Listeria monocytogenes type profile from PubMLST (pubmlst.org). Mapping each of the isolates to the Listeria monocytogenes EGD-e reference genome discovered 110,342 high quality single nucleotide variants (hqSNVs). The presence of genes related to capsular glycan, cell wall/ capsular LTP, biotin biosynthesis, and carbohydrate metabolism associated with amino sugars such as chitin were correlated with biofilm formation. Objective 3: (90% Accomplished) Technological advancements have enabled the addition of probiotics to many types of foods. However, maintaining the viability of probiotic organisms can be a challenge. In our previous studies, a spray-dried formulation was prepared by microencapsulating a combination of probiotic organisms (1:1), Bacillus animalis subsp. lactis ATCC27536 and L. acidophilus ATCC4356, in a conjugated whey protein hydrolysate (WPH) matrix. In the current study, a 'probiotic-butter' was developed by incorporating the conjugated-WPH encapsulated probiotics. Butter was prepared using heavy whipping cream (36% fat), and the butter grains were inoculated with the spray-dried formulation (probiotic population; 8.98±0.02 log CFU/g) at 0.1 or 1%. For salted butter, 1.5% salt was added to the mixture prior to working. Survival of the probiotic bacteria in the 'probiotic butter' was determined immediately after preparation using MRS agar with 0.05% L-cysteine incubated at 37°C in anaerobic jars with gas packs. Triplicate trials were conducted, with analysis of variance used to compare means. The study revealed comparable probiotic counts in salted as well as unsalted butter (p>0.05). The mean counts observed in unsalted 'probiotic butter' were 5.13±0.05 and 8.22±0.02 log CFU/g at 0.1% and 1% inoculum dosages, respectively, whereas in salted 'probiotic butter', the mean counts were 5.09±0.04 and 8.13±0.01 log CFU/g at 0.1% and 1%, respectively. Samples are currently being assessed to monitor survival during storage.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Neha, N., Anand, S. K. 2019. Entrapment of Listeria cells within air-cells of ice cream mix matrix may lead to heat-injured cells. Journal of Dairy Science. 102:9721-9726.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Singh, N., Anand, S., Kraus, B., and Sutariya, S. 2020. Recovery potential of heat-injured cells of Listeria innocua under product abuse conditions vs. simulated gastro-intestinal fluids- a proof of concept study. Journal of Dairy Science. (accepted).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Singh N., Anand S., Gonzalez J. and Kraus B. 2020. Comparing some genetic determinants associated with colonization of Listeria isolates within dairy plant environment. ADSA Virtual Meeting. June 22-24. #M51.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Singh N., Anand S. and Kraus B. 2020. Influence of cell surface properties on the adhesion potential of environmental Listeria isolates to dairy floors. ADSA Virtual Meeting. June 22-24. #T39.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Jha S., Singh N. and Anand S. 2020. Types of Bacillus endospores in milk protein concentrate and milk protein isolate. ADSA Virtual Meeting. June 22-24. #W35.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Minj S. and Anand S. 2020. Developing probiotic butter by incorporating encapsulated probiotic organisms. ADSA Virtual Meeting. June 22-24. #W34.
- Type:
Other
Status:
Other
Year Published:
2020
Citation:
Anand, S. 2020. Enhanced attributes of a conjugated whey proteins in terms of bioactivity, functionality, and as an encapsulant for probiotics. Webinar, Midwest Dairy Foods Research Center. Apr 24, 2020.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Verma, P., Singh, N., and Anand, S. 2020. Competitive exclusion study demonstrates Bacillus subtilis as a predominant constitutive microorganism of reverse osmosis membrane biofilms. Journal of Dairy Science. (accepted).
- Type:
Journal Articles
Status:
Accepted
Year Published:
2020
Citation:
Verma, P., and Anand, S. 2020. Antimicrobial activity may lead to the predominance of Bacillus subtilis within the constitutive microflora of a whey RO membrane biofilm. Journal of Dairy Science. (accepted).
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:The target audience for these projects included dairy processors, dairy equipment manufacturing companies, and dairy separation membrane users. The research outcomes were also shared with the industry personnel and academia, in addition to inclusion in the teaching and instructional content for undergraduate and graduate teaching in the Dairy and Food Science Department. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities to train 1 Post Doc, 4 Ph.D., and 2 MS, and 3 undergraduate students on advanced microbiological and dairy processing techniques. All these students were mentored on day-to-day basis by providing them opportunities to work with the faculty advisor. Belma Husic, undergraduate, 2018. 'Hydrodynamic Cavitation for High Heat Resistant Spore (HHRS) Control in Dairy Industry'. Capstone research project to complete the requirements of Human Biology/ Pre-Medicine major. Dani Rinehart, 2019, (BS Animal Science/ Biotechnology, Oklahoma State University 'Genomic forensics for improving food safety.' Funding: Summer training program in precision livestock production, USDA, REEU undergraduate training program. Maggie Becher, undergraduate, 2018. 'Conducting risk assessment study for Staphylococcus aureus in dairy products.' Funding: MDFRC ongoing research grant plus Schreiber Foods ($1,650). To meet the requirements of Honors College Independent Study. J. F. Nelson Award for undergraduate research ($2,500). Students were also provided professional development opportunities by supporting their travel and participation in regional and national conferences and professional meetings. Neha, N. (doctoral), Awasti, N. (doctoral), Minj, S. (doctoral), Verma, P. (Master's) Chaudhary, P. (Master's) got an opportunity to participate and present at the American Dairy Science Association (ADSA) Annual meeting and Conference at Cincinnati, OH June 23-26, 2019. Neha, N. (doctoral), Minj, S. (doctoral), Verma, P. (Master's). (Master's) got an opportunity to participate and present at the Midwest Dairy Association (MDA) Annual meeting at St. Paul MN, July 29-30, 2019. How have the results been disseminated to communities of interest?The results have been disseminated to the dairy industry and dairy farmers through Midwest Dairy Center meetings, the American Dairy Science Association national meeting, and the National Dairy Council and US Dairy Export Council national spore symposium. In all, three peer reviewed research articles, nine conference and workshop presentations, and two invited talks were delivered. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To improve microbial quality and shelf life of milk and dairy products Task 1.1: To identify quorum inhibitor based anti-biofilm molecules to develop new generation membrane-biofilm cleaners for the dairy industry. This project will be completed this year. Constitutive microflora from dairy separation membranes will be screened for antimicrobial activity. Task 1.2: To study biofilm formation on polished and unpolished stainless steel contact surfaces in dairy industry. This new project has started this year. Different contact surfaces will be studied for surface characteristics and biofilm formation by common spore formers. Objective 2. To improve microbial safety of dairy processes Task 2.1: To control persistent Listeria in dairy processing environment by evaluating resident strains for disinfectant tolerance Studies will be continued to evaluate the resident nature of persistent strains using sequencing techniques. Objective 3. To develop novel dairy products containing probiotics. Task 3.1: To develop a spray-dried health formulation based on whey protein hydrolysate and probiotics encapsulation. Studies will be continued to use the spray dried whey protein hydrolysate conjugate for milk based product development.
Impacts
What was accomplished under these goals?
Objective 1: To improve microbial quality and shelf life of milk and dairy products. (85% Accomplished) Study 1: We hypothesized that combining optimized raw milk holding conditions (T1) and hydrodynamic cavitation (T2) could reduce counts of cells and spores of sporeformering bacteria in spray dried milk powder. Pilot-scale challenge studies were conducted by spiking raw skim milk with approx. 4.0 log cfu/mL vegetative cells and 2.0 log cfu/mL spores of Bacillus licheniformis. Spiked milk samples were then subject to the following treatments: T1, raw milk holding at 4°C for 24 h; T2, raw milk holding at 4°C for 24 h combined with 2 pass hydrodynamic cavitation; and control, raw milk holding at 10°C for 4 h and then at 7°C for up to 72 h (PMO based conditions). Spiked raw milk from the 2 treatments and control were then HTST pasteurized (73°C for 15 s), evaporated, and spray dried to obtain skim milk powders. Final counts of vegetative cells and spores were significantly different (P <0.05) with respect to each other: T1 (0.58 ± 0.04, 1.82 ± 0.05 log cfu/g), T2 (0.33 ± 0.27, 1.49 ± 0.07 log cfu/g), and control (2.74 ± 0.03 and 1.03 ± 0.06 log cfu/g). Our results demonstrate that combining cavitation with optimized raw milk holding conditions (T2) reduced counts of both vegetative cells and spores in spray dried milk powder. Study 2: To understand the microbial interactions within membrane biofilms, emergence of microbial dominance was studied. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. Resuscitation was done in Tryptic Soy Broth (TSB) at 37°C, followed by plating on Tryptic Soy Agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were subsequently identified using MALDI-TOF as Bacillus subtilis, B. licheniformis, Exiguobacterium aurantiacum, and Acinetobacter radioresistens. A competitive exclusion study established the predominance of B. subtilis with a mean log counts of 6.73 ± 0.23 cfu/ mL. Bacillus licheniformis, competed equally with B. subtilis, while not with others. Study 3: Current cleaning and sanitation protocols in milk processing facilities may be ineffective and result in the formation of resilient multispecies biofilms. Antimicrobial activity of B. subtilis as a cause of its predominance in membrane biofilms was investigated. B. subtilis was incubated in TSB at 37°C, and microfiltered to prepare cell-free extracts (CFEs) at 8, 10, 12, 14, 16 and 18h intervals. CFEs were freeze-dried and then tested using an agar-well assay. Results indicated the 12 h CFE of B. subtilis had the highest antimicrobial activity of against Exiguobacterium sp., E. auranticum, A. radioresistens, Listeria monocytogenes, and Bacillus cereus. On treating with proteinase K, the CFE completely lost its antimicrobial activity, establishing the antimicrobial agent to be a proteinaceous compound. Analysis revealed the total crude protein in CFE to be 51% (wt/wt), with glutamic acid (13% wt/wt) a major constituent. The CFE was thermally stable to exposure to the common temperature used for sanitizer applications (23.8°C for 5 and 10 min). An antimicrobial compound was demonstrated to be produced by B. subtilis. Objective 2: To improve microbial safety of dairy processes. (75% Accomplished) Study 1: Serving practices in nursing homes may result in temperature abuse of ice cream before consumption. Presence of even low numbers of injured cells may pose a risk, due to their potential to recover, especially in immunocompromised patients. This investigation was conducted to evaluate injured cell recovery under temperature abuse conditions and on exposure to gastrointestinal (GI) fluids. Based on our previous studies, ice cream mix samples (42% TS) spiked with 4.54 log cfu/gram of Listeria innocua, were lab pasteurized (69°C for 30 min). Heat-injured cells were recovered in Buffered Listeria Enrichment Broth (BLEB), followed by isolation on Modified Oxford Medium (MOX). Ice cream mix samples containing injured cells were followed through overnight aging (7°C), freezing (−4°C), and overnight hardening (−40°C) steps. To simulate serving practices, samples were held for 12 h at 4.4°C, followed by 30 min holding at room temperature (22°C) (identified as the first cycle of temperature abuse). In all, the samples were exposed to 3 such consecutive cycles. At the end of each cycle, direct plating was done on MOX to detect any viable cells. In parallel, the ice cream samples containing injured cells were mixed (1:1) with simulated gastric fluids (pH 1.0 and 2.0) and were held at 37°C in a shaker incubator. Samples were drawn at 15, 30, and 60 min intervals. To study the effect of sequential transit through simulated intestinal fluid, 2 mL of gastric fluid and ice cream (1:1) from gastric fluid experiments were added to 50 mL of simulated intestinal fluid (pH 6.8) and held at 37°C, as above. Samples were drawn at 30, and 360 min intervals. To ascertain the recovery of any injured cells, samples were direct plated on MOX. The temperature abuse or GI fluid exposure studies did not result in recovery of injured cells in the ice cream samples. However, it was also observed that the injured cells were not eliminated during exposure to gastric fluid. Objective 3: To develop novel dairy products containing probiotics. 50% Accomplished Study 1: Whey protein concentrates, isolates, and hydrolysates were screened for bioactivity (antimicrobial activity by agar well assay, antioxidant activity by ABTS+ radical assay, and antihypertensive activity by ACE inhibition assay). The hydrolysates showed significantly (P < 0.05) higher bioactivity (10.6 ± 0.33mm antimicrobial, 766.72 ±13.3 TEAC μmol/L antioxidant, and 67.52 ± 0.2% antihypertensive), compared to the concentrate (8.6 ±0.33mm, 373.3 ± 21.5 TEAC μmol/L, and 60.8 ± 0.1%) and isolate (9.3± 0.33mm, 426.9 ± 42.0 TEAC μmol/L, and 62.9 ± 0.07%). Based on the highest bioactivity, hydrolysate WPH10 was selected for conjugation with maltodextrin. The conjugated WPH10 solution demonstrated higher antimicrobial (17.16 ± 0.33mm) and antioxidant activity (1044.37 ± 39.1TEAC μmol/L) (P < 0.05), whereas a slight decrease in the antihypertensive activity (65.4 ± 0.2%) was observed, as compared with WPH10 alone. Two liters of conjugated solution of WPH10 was spray dried in a Niro drier, with inlet and outlet temperatures of 200 and 90 ± 5°C, respectively, and then freeze-dried at −80°C under 50 mTorr vacuum. The spray dried conjugated-WPH10 exhibited even higher antimicrobial (18.5 ± 0.57mm) and antioxidant activity (1268.89 ± 41.9 TEAC μmol/L) (P < 0.05), while retaining the antihypertensive activity (65.6 ± 0.3%) (P > 0.05). Study 2: We developed a spray dried health formulation by encapsulating probiotics in a conjugated whey protein matrix. Probiotic cultures Bifidobacterium animalis ssp. lactis ATCC27536 and Lactobacillus acidophilus ATCC4356 were added in the WPH10 conjugated solution at ratio of 1:1, at a spiking level of log10 cfu/mL. The mixture was spray dried in a 2-L batch using a Niro drier as described above. The dried formulation was stored in airtight bottles for 10 weeks at 4, 25, and −18°C, to monitor cell viability, moisture, and functionality. Mean probiotic counts in the conjugated WPH10 matrix before and after spray drying were 10.37 log cfu/mL and 8.50 log cfu/g, respectively, and were maintained for at least 4 weeks under all 3 storage conditions. Such formulation can be applied in functional foods to harness their enhanced health benefits and functionality.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2019
Citation:
Neha, N., Anand, S. K. 2019. Entrapment of Listeria cells within air-cells of ice cream mix matrix may lead to heat-injured cells. Journal of Dairy Science. (accepted)
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Awasti, N., S. Anand, and Djira, G. 2019. Sporulating behavior of Bacillus licheniformis strains influences their population dynamics during raw milk holding. Journal of Dairy Science.102:6001-6012.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Minj, S., Anand, S. K. 2019. Microencapsulation of probiotic organisms within a conjugated whey protein hydrolysate matrix. American Dairy Science Association ADSA Annual Meeting. Cincinnati, OH. June 24.
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
Neha, N., Anand, S. K., Djira, G., Kraus, B., Sutariya, S. 2018. Listeria cross contamination levels in raw ice cream mix can serve as a predictor of their presence as heat-injured cells. Journal of Dairy Science. 101:9659-9669.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Minj, S., Anand, S. K. 2019. A conjugated whey protein hydrolysate demonstrates enhanced bioactive attributes. American Dairy Science Association Annual Meeting. Cincinnati, OH. June 24.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Verma, P., Anand, S. K. 2019. ADSA, A natural antimicrobial from Bacillus subtilis, a predominant constituent of membrane biofilms. American Dairy Science Association Annual Meeting. Cincinnati, OH. June 24.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Verma, P., Singh, N., Anand, S. K. 2019. Competitive exclusion study demonstrates Bacillus subtilis as a predominant constitutive microorganism of RO membrane biofilms. American Dairy Science Association ADSA Annual Meeting. Cincinnati, OH. June 24.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Minj, S., Anand, S. K. 2019. Developing a dairy based health formulation by combining the bioactive properties of whey protein hydrolysates and probiotic organisms. American Dairy Science Association ADSA Annual Meeting. Cincinnati, OH. June 24.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Awasti, N., Anand, S. K. 2019. Manufacturing low spore count skim milk powder by controlling raw milk holding conditions- a pilot scale trial. American Dairy Science Association ADSA Annual Meeting. Cincinnati, OH. June 24.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Awasti, N., Chaudhary, P., Anand, S. K. 2019. Manufacturing low spore count skim milk powders by combining optimized raw milk holding conditions and hydrodynamic cavitation. American Dairy Science Association ADSA Annual Meeting. Cincinnati, OH. June 24.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Singh, N., Anand, S. K. 2019. Recovery potential of heat-injured cells of Listeria under ice cream temperature abuse conditions vs. simulated gastrointestinal fluids. American Dairy Science Association ADSA Annual Meeting. Cincinnati, OH. June 24.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Becher, M., Anand, S. K. (Advisor), Singh, N., Becher, M. 2019. Effect of varying salt concentrations on S. aureus growth in a nonfat dry milk slurry. Dairy and Food Science Department, SDSU, Brookings, SD, United States. April 9.
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
Sanjeev, A. 2019. Strategies for minimizing sporeformers and spores during milk powder processing. Spore Symposium. ADSA Annual meeting, Cincinnati, OH. June 24.
- Type:
Other
Status:
Other
Year Published:
2019
Citation:
Sanjeev, A. 2019. Techniques for producing low spore dried products. NCCIA 2019 Annual Conference. Rochester, MN. Oct 8-10.
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience: The target audience for these projects included dairy processors, dairy equipment manufacturing companies, and dairy separation membrane users in the dairy industry. The research outcomes were also shared with the industry personnel, and academia, in addition to inclusion in the teaching and instructional content for undergraduate and graduate teaching in the Dairy and Food Science Department. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?The project provided opportunities to train 4 undergraduates, 3 Ph.D., and 2 MS students on advanced microbiological and dairy processing techniques. All these students were mentored on day-to-day basis by providing them opportunities to work with the faculty advisor. Belma Husic, undergraduate, 2018. 'Hydrodynamic Cavitation for High Heat Resistant Spore (HHRS) Control in Dairy Industry'. Capstone research project to complete the requirements of Human Biology/ Pre-Medicine major. Jene Martin, undergraduate, 2018, (Microbiology Major, University of California, San Diego), 'Genomic forensics for improving food safety.' Funding: Summer training program in precision livestock production, USDA, REEU undergraduate training program. Marie Zander, undergraduate, 2018. 'Standardizing a non-thermal process for controlling spore formers' Funding: DMI ongoing research grant. To meet the requirements of Honors College Independent Study. J. F. Nelson Award 2018 for undergraduate research. Maggie Becher, undergraduate, 2018. 'Conducting risk assessment study for Staphylococcus aureus in dairy products.' Funding: MDFRC ongoing research grant plus Schreiber Foods ($1,650). To meet the requirements of Honors College Independent Study. J. F. Nelson Award for undergraduate research ($2,500). Students were also provided professional development opportunities by supporting their travel and participation in regional and national conferences and professional meetings. Neha, N. (doctoral), Awasti, N. (doctoral), Chaudhary, P. (Master's) got an opportunity to participate and present at the American Dairy Science Association (ADSA) Annual meeting and Conference at Knoxville, TN, June 24-27, 2018. Neha, N. (doctoral), Awasti, N. (doctoral), Minj, S. (doctoral), Verma, P. (Master's), Chaudhary, P. (Master's) got an opportunity to participate and present at the Midwest Dairy Association (MDA) Annual meeting at St. Paul MN, July 31-Aug 01, 2018. How have the results been disseminated to communities of interest?The results have been disseminated to the dairy industry and dairy farmers through Midwest Dairy Center meetings, American Dairy Science Association national meeting, and National Dairy Council and US Dairy Export Council national spore symposium. Our Listeria research was also featured in the national media and covered by prominent science dissemination platforms such as Food Safety News and Food Quality and Safety Magazine. In all, six peer reviewed research articles, one review paper, one magazine article, nine conference and workshop presentations, and five invited talks were delivered. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: To improve microbial quality and shelf life of milk and dairy products Task 1.1: To understand sporulation behavior of common sporeformers during milk powder manufacture This project will be completed this year by conducting the pilot scale trials, and the consolidated report will be submitted to the funding agency. Task 1.2: To apply non-thermal technologies, such as cavitation, to control common dairy sporeformers This project will be completed this year by conducting the pilot scale trials, and the consolidated report will be submitted to the funding agency. Task 1.3: To identify quorum inhibitor based anti-biofilm molecules for developing new generation membrane-biofilm cleaners for the dairy industry. A new project has been initiated under this objective. Constitutive microflora from dairy separation membranes will be screened for antimicrobial activity. Goal 2. To improve microbial safety of dairy processes Objective 2.1: To control dairy pathogens such as Listeria by risk analysis, through response surface models. This project will be completed this year by conducting the pilot scale trails, and the consolidated report will be submitted to the funding agency. Task 2.2: To control persistent Listeria in dairy processing environment by evaluating resident strains for disinfectant tolerance A new project has been initiated under this objective. Studies will be conducted on evaluating the resident nature of persistent strains using sequencing techniques. Objective 3. To develop novel dairy products containing probiotics. Task 3.1: To develop a spray-dried health formulation based on whey protein hydrolysate and probiotics encapsulation. We plan to use the spray dried whey protein hydrolysate conjugate for milk based product development.
Impacts
What was accomplished under these goals?
Objective 1: To improve microbial quality and shelf life of milk and dairy products. 75% Accomplished To understand the role of strain variability, population dynamics of two strains of Bacillus licheniformis, ATCC 6634 and 14580, were modeled as a function of temperature (4.0?, 8°, and 12.0? C) and duration (0 - 72 h) using regression analysis. Based on the initial spiking of vegetative cells (approx. 4.0 log cfu/mL) and spores (approx. 2.0 log cfu/mL), six regression equations elucidating B. licheniformis growth behavior during raw milk holding at low temperatures were obtained based on y = β0 + β1x1 + β2x2 + β11x11 + β22x22 + β12x12 + ε (2), where, x11 and x22 are quadratic terms and x12 is an interaction between temperature and duration.. Contour plots were developed to determine the temperature-duration combinations that kept the population changes to less than 1.0 log. In the vegetative cell spiking study of B. licheniformis ATCC 6634 (S1), cell population changes remained below 1.0 log up to 72 h at 8? C. For B. licheniformis ATCC 14580 (S2), a 1.0 log shift was observed only after 80 h at 8°C, These results showed a greater multiplication potential of B. licheniformis ATCC 6634 (S1) as compared to B. licheniformis ATCC 14580 (S2). As S2 was a readily sporulating strain, the vegetative spiking study showed spore formation at different storage temperatures. An evidence of some parallel germination was observed for this strain at 8? C or higher, when raw milk samples were spiked with spores. The experimental values obtained for sporeformers and spore counts compared well with contour plots generated values. Overall, for raw milk samples containing the low sporulating strain (B. licheniformis ATCC 6634), the contour plots suggest that milk could be held safely at 8?C or below for up to 72 h. In the case of the readily sporulating strain (B. licheniformis ATCC 14580), raw milk could be held safely at 8?C for up to 80 h. Sporulation behavior, germination and multiplication ability, strain variability, and temperature and duration of holding raw milk influenced the population dynamics of Bacillus species. However, in the presence of equivalent numbers of both types of sporulating strains in raw milk, despite of strain variability, holding milk at 8? C for not more than 72 h would keep any cell population changes below 1.0 log. In addition, under these storage conditions, the population would remain as vegetative cells that are easily inactivated by pasteurization. The contour plots, so generated, would help predict the population shifts and define optimum holding conditions for raw milk before further processing. Objective 2: To improve microbial safety of dairy processes. 50% Accomplished Listeriosis is a life-threatening infection caused by foods contaminated with Listeria monocytogenes. Some of the major ice cream recalls in recent years reaffirm the ability of this food borne pathogen to survive in diverse dairy processing environments and cause cross contamination. Inspection reports revealed lapses in implementing adequate hygienic practices resulted in Listeria persistence in the processing environment, leading to cross contamination of ice cream. The higher levels of cross contamination of raw ice cream mix may result in some heat-injured cells when exposed to minimum heat treatment (69°C for 30 min). These heat-injured cells could later recover under abused storage and handling conditions, and pose a health risk. Evidence about the presence of injured cells in ice cream mix may thus prove useful to establish the overall Listeria risk, which is the aim of this study. Challenge studies were conducted to evaluate the dose dependent presence of heat-injured cells of Listeria. Ice cream mix formulations of four different types (total solids % 36, 40, 42 and 45) were inoculated with 2.0, 3.0, and 4.0 log cfu/g levels of Listeria innocua (an established surrogate). The dose levels were selected based on a likely cross contamination on the raw side from environmental Listeria, especially due to their resident nature and growth in harborage sites. The samples were exposed to minimum heat treatment (69°C for 30 min), and the survivors, including heat-injured cells, were enumerated using standard protocols. A binary logistic regression model was fitted to evaluate the severity of risk. The influence of total solids, water activity, and pH variability on Listeria survival were also studied. The enrichment protocol, using Buffered Listeria Enrichment Broth (BLEB) followed by plating on Modified Oxford Agar and Rapid L' mono medium, revealed the random presence of heat-injured cells in BLEB only at the highest dose level of 4+ logs. Any potential risk from heat-injured cells was thus limited only to the highest levels of cross contamination, irrespective of the type of the mix. Significantly, none of the pasteurized ice cream mix samples supported the recovery of any heat-injured cells of Listeria during 72h holding at 7°C, even at the highest dose level of 4+ logs, under the conditions of experimentation. The level of cross contamination (dose) emerged as a predictor of the potential presence of heat-injured cells of Listeria exposed to minimum pasteurization treatment. Objective 3: To develop novel dairy products containing probiotics. 25% Accomplished Proteins and peptides extracted from whey protein and hydrolysates through enzymatic hydrolysis exhibit biological properties and confer health benefits to the consumers. In addition, studies have shown that protein ingredients produced from conjugation of protein with maltodextrin have potential to offer greater functionality in food applications, but the effects of conjugation on biological properties has yet to be determined. In our current investigation, samples of whey protein concentrate (WPC 80), whey protein isolate (WPI 90), and whey protein hydrolysates (WPH10, WPH15 and WPH20) were screened for their biological properties (antimicrobial, antioxidant and antihypertensive properties). The antimicrobial activity was checked by agar well diffusion assay, while the antioxidant activity was determined by ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) assay. The antihypertensive activity was tested by in vitro angiotensin-converting-enzyme inhibitor (ACE inhibitor) activity. Results of our work showed that whey protein hydrolysates exhibited higher biological activities compared to intact whey protein samples. For antimicrobial activity, the average inhibitory zone size was 10.4mm for hydrolysates, as compared to 8.6mm for the intact protein. In the case of antioxidant activity the average values were 866.5 TEAC µmol/L and 375.3 TEAC µmol/L, respectively. In case of ACE inhibition, the values observed were 67.5% and 60.8%, respectively. The WPH10 material (that showed the highest biological properties) was successfully conjugated with maltodextrin, and a thermally stable conjugated solution was obtained. The extent of conjugation was determined by recording the change in pH at regular intervals during heating, and conjugation was confirmed by a sharp decrease in pH of the conjugated solution from 8.2 to 6.7 at the end of heating period. The conjugated solution was spray dried and freeze-dried and the effect of conjugation on the modifications of the biological properties was assessed. There was no difference in the antihypertensive property. However, conjugation resulted in an increase in the antimicrobial activity (shown by an increase in inhibitory zone from 10.4mm to 19.0mm), and antioxidant properties (that increased to 1324.13 TEAC µmol/L). Hence, such conjugates can be potentially employed as ingredients in food industries and for the development of functional foods.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Awasti, N., Anand, S. K., Djira, G., ADSA Annual Conference, "Sporulating behavior influences the population dynamics of sporeformers during raw milk holding." Knoxville, TN, United States. (2018).
J. Dairy Sci. Vol. 101, Suppl. 2. M114. P 47
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Neha, N., Anand, S. K., ADSA Annual Conference, "Tracking Listeria survival at different stages of ice cream manufacture." Knoxville, TN, United States. (2018).
J. Dairy Sci. Vol. 101, Suppl. 2. No.22. p 123
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Sim, J. Y., Martinez Monteagudo, S. I., Anand, S. K., Gamma Sigma Delta, Honors Society of Agriculture, "Hydrodynamic cavitation in dairy manufacturing: Characterization and microbial impact on skim milk concentrate.," South Dakota State University, Brookings, SD, United States. (2018).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Neha, N., Anand, S. K., Djira, G., Kraus, B., Sutariya, S., Gamma Sigma Delta, Honors Society of Agriculture, "Level of Listeria cross contamination in ice cream mix can serve as a predictor of its overall risk from injured cells. Graduate Poster Competition." South Dakota State University, Brookings, SD, United States. (2018).
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Awasti, N., Suliman, R., Anand, S. K., Djira, G., Gamma Sigma Delta, Honors Society of Agriculture, "Population dynamics of a common dairy sporeformer, Bacillus licheniformis, in spiked raw milk samples stored at low temperatures.," South Dakota State University, Brookings, SD, United States. (2018).
- Type:
Other
Status:
Other
Year Published:
2018
Citation:
Anand, S. K., MDFRC Annual Research Meeting, "Assessing implications of weldments and polished stainless steel surfaces in supporting spore former biofilms in dairy plants," MDFRC, University of Minnesota, St. Paul, MN, United States. (July 31, 2018).
A new grant proposal was presented for peer review and funding.
- Type:
Other
Status:
Other
Year Published:
2018
Citation:
Anand, S. K., MDFRC Annual Research Meeting, "Whey Protein Hydrolysate-Probiotic Encapsulants," MDFRC, University of Minnesota, St. Paul, MN, United States. (July 30, 2018).
- Type:
Other
Status:
Other
Year Published:
2018
Citation:
Anand, S. K., MDFRC Research Planning Meeting, "Listeria and ice cream-A new perspective," MDFRC, Brookings, SD, United States. (May 8, 2018).
- Type:
Other
Status:
Other
Year Published:
2018
Citation:
Anand, S. K., 2018 U.S. Dairy Industry Spore Symposium, "Biofilm removal in membrane processing," US Dairy Export Council and National Dairy Council, Morro Bay, California, Morro Bay, CA, United States. (March 2, 2018).
- Type:
Other
Status:
Other
Year Published:
2018
Citation:
Anand, S. K., 2018 U.S. Dairy Industry Spore Symposium, "Population dynamics of sporeformers is influenced by low and high sporulation strains.," US Dairy Export Council and National Dairy Council, Morro Bay, California, Morro Bay, CA, United States. (March 2, 2018).
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Jindal, S., Anand, S. K. (2018). Comparison of adhesion characteristics of common dairy sporeformers and their spores on stainless steel and modified contact surfaces. Journal of Dairy Science, 101(7), 5799-5808.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Marka, S., Anand, S. K. (2018). Feed substrates influence biofilm formation on reverse osmosis (RO) membranes and their cleaning efficiency. Journal of Dairy Science, 101(1), 84-95.
(Selected as Editor's choice paper in the January issue of the Journal of Dairy Science as an Editors Choice ).
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2018
Citation:
Neha, N., Anand, S. K., Djira, G., Kraus, B., Sutariya, S. (2018). Listeria cross contamination levels in raw ice cream mix can serve as a predictor of their presence as heat-injured cells. Journal of Dairy Science. (in press)
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Gupta, S., Anand, S. K. (2018). Induction of pitting corrosion on stainless steel (grades 304 and 316) used in dairy industry by biofilms of common sporeformers. International Journal of Dairy Technology, 71(2), 519-531.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Jindal, S., Anand, S. K., Metzger, L., Amamcharla, J. (2018). A comparison of biofilm development on stainless steel and modified surface (Ni-P-PTEF) plate heat exchangers during a 17h milk pasteurization run. Journal of Dairy Science, 101, 2921-2926.
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Liu, D. Z., Jindal, S., Amamcharla, J., Anand, S. K., Metzger, L. (2017). Evaluation of a sol-gel based stainless steel surface modification to reduce fouling and biofilm formation during pasteurization of milk. Journal of Dairy Science, 100(4), 2577-2581.
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Anand, S. K., Neha, N. (2018). More Robust Risk Assessment. Food Quality and safety, 4(25): 15-16. Part of an article Pathogen Patrol: Learning on Listeria, a magazine article authored by Linda L. Leake.
- Type:
Other
Status:
Published
Year Published:
2017
Citation:
Adamski, A., Bawa, D., Fernandez, N. G., Anand, S. K. (2017). Reported incidences of dairy pathogens. A review. MDFRC (Midwest Dairy Foods Research Center). Pages 1-63
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Sim, J. Y., Martinez Monteagudo, S. I., Anand, S. K., ADSA Annual Conference, "Development of a Continuous Cavitation-Assisted Thermal Treatment for Skim Milk Concentrate: Process Characterization and Microbial Efficiency," Knoxville, TN, United States. (2018).
J. Dairy Sci. Vol. 101, Suppl. 2. No.467. p 400
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Neha, N., Anand, S. K., B. K., S. S., ADSA Annual Conference, "Effect of freezing and hardening on injured versus intact cells Listeria in ice cream mix.," Knoxville, TN, United States. (2018).. Dairy Sci. Vol. 101, Suppl. 2. M117. P 48
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Chaudhary, P., Anand, S. K., Martinez Monteagudo, S. I., ADSA Annual Conference, "Feasibility of hydrodynamic cavitation in-line with HTST pasteurization for inactivating sporeformers and spores in skim milk.," Knoxville, TN, United States. (2018).
J. Dairy Sci. Vol. 101, Suppl. 2. M115. P 47
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Awasti, N., Anand, S. K., ADSA Annual Conference, "Ratiometric fluorescence spectroscopyA novel technique for rapid detection of bacterial endospores." Knoxville, TN, United States. (2018).
J. Dairy Sci. Vol. 101, Suppl. 2. M8. P 9
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