Progress 08/01/24 to 07/31/25
Outputs
Target Audience:The target audiences include ultrasound equipment manufacturers, the protein fermentation sector, the plant-based protein industry, and the broader food science community. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?A graduate student has been trained in fermentation and protein characterization. She has also received training in flavor analysis using GC-MS and in statistical data interpretation. How have the results been disseminated to communities of interest?Part of these results have been presented in scientific conferences such asAOCS Annual meeting & Expo. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: The upcoming work will focus on selecting the most effective lactic acid bacterial strain based on its efficiency in off-flavor removal and protein precipitation. Initial screenings have already been completed for some strains, while analyses for the remaining strains are still in progress. Once all strains have been evaluated, the most efficient strain will be identified based on its ability to remove off-notes generated from both volatile and non-volatile components, as well as its protein precipitation efficacy. Objective 2: The protein isolates obtained from Objective 1 will be compared with those produced using traditional methods to assess overall performance. The research will emphasize analyzing changes in molecular structure using techniques such as SDS-PAGE, FTIR, and hydrophobicity testing, along with evaluating functional attributes such as solubility, foaming capacity, and emulsifying ability. In addition, anti-nutritional factors and nutritive components will be assessed, with particular focus on amino acid composition, phenolic compounds, in-vitro digestibility, phytic acid, and trypsin inhibitors. Furthermore, the textural properties of vegan plant-based meat and cheese produced using the selected protein isolates will be compared. This comparison will help identify the most efficient method for producing nutritious, high-quality, and functional plant proteins suitable for a range of applications. By the next reporting period, most of the tasks under Objective 2 are expected to be completed, thereby contributing to the overall goal of developing a novel plant protein ingredient.
Impacts
What was accomplished under these goals?
Objective 1. Optimize the ultrasound process to release protein in the extracting solution (water) and identify the best lactic acid bacteria strain(s) based on protein precipitation and off-notes removal efficiency. The ultrasound-assisted extraction (UAE) was optimized for both chickpea and pea flours. The extraction was performed by mixing flour with distilled water at a 1:10 (w/v) ratio and subjecting it to ultrasound treatment for 2, 4, 6, 8, and 10 minutes using a 50% duty cycle (5 seconds on and off). All samples were tested for solubility using the Bradford method. Overall, ultrasound treatment for 4 minutes was found to be effective for retaining proteins from both chickpea and pea samples. The isoelectric precipitation (IEP) of pea and chickpea proteins was optimized to pH 4.5 based on solubility tests using the Bradford method. The alkali-extracted chickpea samples showed lipoxygenase activity (3.27 ± 0.15 U/g) and lipase content (0.16 ± 0.00 U/g). These results confirm the presence of oxidative enzymes responsible for producing off-flavor compounds in chickpea samples. Key off-flavor compounds identified through GC-MS analysis included hexanal (269.4 ± 17.74 ppb), octanal (139.8 ± 2.72 ppb), heptanal (13.6 ± 0.31 ppb), trans-2-hexenal (5.3 ± 0.58 ppb), and 1-octanol (7.18 ± 0.16 ppb). These findings indicate the presence of aldehydes and alcohols that impart characteristic grassy and beany off-flavors in chickpea samples. Five lactic acid bacteria strains (pellets)--Pediococcus pentosaceus, Lactobacillus casei, Lactobacillus fermentum, Lactococcus lactis, and Pediococcus acidilactici--were purchased from the USDA-ARS culture collection and stored at −80oCfor analysis. The strains were revived in MRS broth following ARS protocols and incubated at 37oC. Fresh subcultures were prepared from the revived strains to ensure consistency across batches. To determine the growth patterns of individual strains and the optimum inoculation time, 24-hour growth curve analyses were conducted. Viable plate count, optical density (OD600), and pH were monitored every 2 hours. This analysis helped identify the ideal inoculation time--at the end of the log phase, when bacterial activity and growth are highest. Based on OD600 measurements, L. fermentum reached the end of the log phase at ~5 hours. For L. casei and P. pentosaceus, the log phase ended at ~8 hours. P. acidilactici transitioned to the stationary phase after ~7 hours, while L. lactis displayed the longest log phase, lasting ~13 hours. For protein extraction, chickpea and pea flours were suspended in distilled water (1:10 w/v), treated with ultrasound, and adjusted to pH 7. An alkali-extracted sample without ultrasound (AE) was also prepared under identical conditions for comparison. The samples were centrifuged at 8,000 rpm for 20 minutes, and the supernatant was collected for bacterial inoculation. Bacterial cultures were grown to a cell concentration of 107-108 CFU/mL. Fresh cultures were prepared until they reached the log phase. Once in the log phase, bacterial cells were harvested, washed, and centrifuged three times with phosphate-buffered saline. Then, 5% of the bacterial culture was inoculated into the protein extract supernatant, and pH, OD600, and viable plate counts were monitored every 2 hours. Fermentation continued until the samples reached their isoelectric point (pH 4.5). Samples were then centrifuged at 8,000 rpm for 20 minutes, washed with distilled water, neutralized to pH 7, freeze-dried, and stored at −4oC for further analysis. In P. pentosaceus-inoculated pea samples, the AE samples attained IEP at 20 hours, while the UAE samples reached IEP at just 10 hours. The purity of fermented samples showed that UAE samples had 90.4% purity, compared to 94.7% for AE samples. Although fermented UAE pea samples had slightly lower purity, they achieved a higher total mass yield (12.48%) compared to AE (10.8%). Total protein recovery was also greater in UAE samples (53.55%) than AE (51.5%). For chickpea, AE samples achieved fermentation pH at 12.5 hours, while UAE samples reached IEP at 12 hours. The fermented UAE samples had a purity of 69.85%, compared to 82.85% in AE samples. However, UAE samples produced a higher total mass yield (17.15%) versus AE (13.6%), with higher total protein recovery (63.02% vs. 59.31%). In L. fermentum-fermented pea samples, UAE samples reached IEP at 10 hours compared to 12 hours for AE. The fermented AE samples showed higher purity (94.3%) compared to UAE (87.4%). Total mass yields were similar between UAE (17.9%) and AE (17.4%). Protein recovery, however, was higher in UAE (53.55%) compared to AE (51.14%). In chickpea samples, both AE and UAE reached IEP at 12 hours. The UAE samples showed lower purity (67.9%) compared to AE (82.3%), but UAE achieved a higher mass yield (16.4% vs. 15.35%) and greater protein recovery (66.49% vs. 64.93%). In L. casei-fermented pea samples, AE reached IEP at 16 hours, while UAE reached IEP at 14 hours. The initial protein concentration in the UAE supernatant was higher (13.48 mg/mL) compared to AE (11.65 mg/mL). By the end of fermentation, protein concentration declined to 1.01 mg/mL in AE and 1.03 mg/mL in UAE. The initial sugar concentration was higher in UAE (8.44 mg/mL) compared to AE (7.96 mg/mL). After fermentation, sugar content decreased to 5.81 mg/mL in UAE and 4.42 mg/mL in AE. The fermented AE samples had higher purity (94.3%) than UAE (86.9%). However, UAE showed higher mass yield (12.9% vs. 11.15%) and higher protein recovery (53.55% vs. 51.15%). In L. casei-fermented chickpea samples, AE reached IEP at 19.5 hours, while UAE achieved IEP at 19 hours. The initial protein concentration was slightly higher in UAE (12.39 mg/mL) compared to AE (12.33 mg/mL). After fermentation, protein content decreased to 0.213 mg/mL in AE and 0.341 mg/mL in UAE. Initial sugar concentration was 8.59 mg/mL in AE and 8.79 mg/mL in UAE. By the end of fermentation, sugar content decreased to 4.22 mg/mL in AE and 4.63 mg/mL in UAE. The purity of UAE samples was 70.1%, compared to 79.55% in AE samples. However, UAE yielded a higher total mass (17.6% vs. 13.5%) and higher protein recovery (64.93% vs. 56.51%).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Lakshmipathy, K., Modupalli, N., Rahman, M. M. (May 26-30, 2025). Lactic acid bacteria as a method to improve off-flavor and enhance the quality of pulse protein. 2025 AOCS Annual meeting & Expo, Portland, OR, USA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2025
Citation:
Lakshmipathy, K., Modupalli, N., Lafontaine, S., & Rahman, M. M. (May 26-30, 2025). Enhance the neutral taste and quality of chickpea protein by integrating acid extraction with conventional methods. 2025 AOCS Annual meeting & Expo, Portland, OR, USA.
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