Location: Dairy and Functional Foods Research
2025 Annual Report
Objectives
Objective 1: Characterize bioactive peptides released from dairy proteins by enzymatic digestion and investigate the effect of protein-carbohydrate interactions on the stability and bioavailability of these peptides in dairy foods. [NP306, C1, PS1C]
Sub-objective 1.A: Production and characterization of bioactive peptides from dairy proteins through enzymatic digestion.
Sub-objective 1.B: Develop encapsulation-based delivery systems to protect and enhance the bioactivity and effectiveness of these peptides.
Objective 2: Identify novel bioactive prebiotic oligosaccharides from plant (fruit and vegetable) and animal (milk) sources in food processing low-value by-products. [NP306, C1, PS1B]
Approach
New bioactive compounds will be developed from dairy proteins, oligosaccharides and plant dietary fiber. Bioactive compounds will include antioxidative peptides from milk and dairy products. We will identify these bioactive peptides released through the enzymatic digestion of dairy proteins and products, and investigate the effect of milk fatty acids and oligosaccharides on the production of these peptides. Novel prebiotics will be characterized and compared with galacto-oligosaccharides derived from lactose and milk oligosaccharides. Pectic oligosaccharide prebiotic activity will be evaluated using animal models, mixed culture fecal fermentation and determining their effects as human bioactive food ingredients. We will develop synbiotics in live microbial systems such as yogurt and with tea products. Oligosaccharide anti-adhesive activity against pathogens will be determined with tissue culture cells. We will determine dietary fiber characteristics in fruit and vegetable processing byproducts.
Progress Report
Objective 1. Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. Following fermentation, two soluble protein fractions were collected, CFS1, which contained mainly whey proteins, and CFS2, which was predominantly caseins, were analyzed for protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2, but CFS2, mainly showed a higher level of ACE-I activity (33.8%). Significant differences were also found in the amounts of d- and l-lactic acid produced by each strain in UHT milk. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation when trying to elicit specific bioactivities.
Objective 2. ß-Galactosidase is currently applied in foods for reduction of lactose but can also be used for its transgalactosylation activity to synthesize galacto-oligosaccharides (GOS) as prebiotics. The ability of GRAS-status Lactobacillus strains to exhibit such activities would benefit consumers given their extensive history with dairy products. The objective of this study was to characterize the production of GOS in 6 Lactobacillus strains for their ability to synthesize GOS in 50 mM sodium phosphate (pH 6.5) with a high lactose concentration of 788.8 mM at 50 °C. Lysates from Lactobacillus delbrueckii ssp. bulgaricus LB11 and YB1 and L. helveticus B548 released the most glucose at 112.8 ± 6.2 mM, 150.4 ± 11.7 mM, and 190.2 ± 12.2 mM, respectively. However, the ratio of free glucose to galactose released by B548 lysate (1.4 ± 0.3) was lower than that of LB11 (2.8 ± 0.6) and YB1 (2.5 ± 0.6) lysates, so the latter strains were screened at different initial lactose concentrations. The GOS yield from YB1 was not dependent on initial lactose concentration, averaging 54.3 ± 0.6% across starting lactose concentrations. However, optimal LB11 transgalatosylation had an initial concentration of 394.4 mM lactose instead of 788.8 mM, resulting in a GOS yield of 56.8% instead of 47.3%. In all cases for LB11, the lysates had greater free glucose, galactose, and GOS yield than whole cells. The ability of 2 Lactobacillus delbrueckii ssp. bulgaricus strains to produce GOS from common lactose-containing ingredients can have a range of applications in the dairy industry. Since many dairy products rely on sweeteners for flavor and texture, B-gal from Lactobacillus bulgaricus strains LB11 and YB1, L. helveticus B548, and Kluyveromyces lactis (KL, Lactozyme® 2600L) were used to determine if transgalactosylation can be driven in reconstituted nonfat dry milk (NDM, 10%, wt/wt) with commercial sweeteners compared to unsweetened NDM at 50 °C for 18 h. Sucrose (14.4%, wt/wt), corn syrup solids (14.4%, wt/wt), or a mixture of sucrose (9.9%, wt/wt) and corn syrup solids (4.5%, wt/wt) were dissolved in NDM (n=3), then heated with stirring for 30 minutes at 70 °C for batch pasteurization. For Lactobacillus strains, whole cells, inoculated at 7 log CFU/mL, or cell lysates were added to the milk and carbohydrate mixtures, or 1.85 U of KL B-gal. The sucrose mixture released the highest glucose from all B-gal preparations, with LB11 lysate releasing the highest (245.57 ± 25.16 mM). High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection was used to visualize the presence of oligosaccharides with degrees of polymerization of 3 to 7. Oligosaccharides generated in NDM + sucrose treated with the YB1 lysate increased the anaerobic growth of Bifidobacterium breve 2141 at 37 °C compared to untreated or KL B-gal treated sucrose mixtures. B-gal treatment of nonfat milk containing sucrose and corn syrup can be a source of prebiotic oligosaccharides, although more work is needed to determine the composition of the oligosaccharides.
Objective 2. Dietary fiber composition from blueberry, strawberry, red beet and carrot residue remaining after commercial processing. The dietary fiber was largely insoluble containing pectin, xyloglucan, arabinoxylan, xylan, ß-glucan and glucomannan polysaccharides, fructose, glucose, xylose, arabinose, galactose, fucose and galacturonic acid free sugars, protein, fat, and anthocyanins (malvidin 3-arabinoside and peonidin 3-glucoside in blueberry and pelargonidin 3-glucoside in strawberry). The pectin was determined to be methyl-esterified, acetylated and feruloylated rhamnogalacturonan I often with a 4,5-unsaturated function at the non-reducing end based on oligosaccharide structural analysis. A random coil shape was observed for pectin associated with strong anthocyanin binding. The structural properties of the blueberry, strawberry and red beet fiber fractions suggest that they can function to thicken and stabilize food systems, and the structure of the pectin fractions indicate strong potential as a prebiotic food ingredient. The structural properties of the carrot pomace fiber fractions suggest that they can function as a low viscosity beverage food ingredient, and the rhamnogalacturonan I structure indicated the potential for anti-viral, immuno-modulatory and prebiotic properties.
Objective 2. Synbiotics were developed from cranberry pomace and corn dried distillers’ grains with solubles. This study further characterized cranberry xyloglucan and pectic oligosaccharide structures from pectinase-treated cranberry pomace and measured the growth and short-chain fatty acid production of 86 Lactobacillus strains using a cranberry oligosaccharide fraction as the carbon source. In addition to arabino-xyloglucan structures, cranberry oligosaccharides included pectic rhamnogalacturonan I which was methyl-esterified, acetylated and contained arabino-galactooligosaccharide side chains and a 4,5-unsaturated function at the non-reducing end. When grown on cranberry oligosaccharides, ten Lactobacillus strains reached a final culture density (change OD) = 0.50 after 24 h incubation at 32 °C, which was comparable to L. plantarum ATCC BAA 793. All strains produced lactic, acetic, and propionic acids, and all but three strains produced butyric acid. This study demonstrated that the ability to metabolize cranberry oligosaccharides is Lactobacillus strain specific, with some strains having the potential to be probiotics, and for the first time showed these ten strains were capable of growth on this carbon source. The novel cranberry pectic and arabinoxyloglucan oligosaccharide structures reported here combined with the Lactobacillus strains that can metabolize cranberry oligosaccharides and produce short-chain fatty acids, have excellent potential as health-promoting synbiotics.
Corn arabinoxylans (AX) and protease treated AX (AXP) were subjected to enzymatic hydrolysis with endoxylanase and arabinofuranosidase to obtain hydrolyzed AX (HAX) and AXP (HAXP), whose ability to promote Bifidobacterium infantis and Bifidobacterium longum growth was investigated. Further, the effect of cross-linked AX on the growth of bifidobacteria was also explored. Bifidobacteria showed the highest growth on AX and AXP, while HAX and HAXP did not have a significant impact on bacterial growth. The laccase cross-linking of AX stimulated the growth of bifidobacteria, possibly due to its gel-like structure which favored the bacteria-substrate (AX) interaction. The laccase-induced cross-linked AX (AXG) and alginate (SA) were used to prepare encapsulating matrices. The ability of AXG-SA matrices to encapsulate and protect probiotic bacteria ( Lactobacillus rhamnosus GG, Streptococcus thermophilus and B. longum ) viability under storage conditions was investigated. The AXG matrices presented the highest encapsulation efficiencies (55–77%) for all three strains, when AXG-SA and SA matrices were compared. Significantly higher levels ( ~7 logs) of L. rhamnosus GG were recovered from AXG and AXG-SA matrices after 28 days of storage under aerobic conditions at 4 °C compared to SA matrices ( ~4 logs). The results indicated that the incorporation of AX into the matrices played a significant role on the survival of encapsulated bacteria during storage, which could be attributed to the stability of the covalent cross-linked network formed during AX gelation that protected bacterial viability. Thus, the matrices based on AXG could be promising materials to encapsulate and protect probiotic bacteria for targeted-delivery to the colon.
Accomplishments
1. Prebiotics produced enzymatically from non-fat milk and sucrose. Commercial prebiotics, such as galacto-oligosaccharides, are food ingredients that promote gut health. These prebiotics are produced by microbial enzymes that require high concentrations of lactose, which requires additional steps to separate the excess lactose from the final products, thereby increasing their cost. ARS researchers at Wyndmoor, Pennsylvania, determined that enzymes from a yogurt-producing bacterium and a yeast can produce prebiotics in nonfat milk containing sucrose at lower concentrations than the high lactose concentrations used previously. The yogurt-producing bacterial prebiotics supported greater growth of a gut health promoting bacterium compared to those produced by the yeast enzyme. The use of a dairy starter culture bacterial strain that efficiently produces potential prebiotics can add health benefits to dairy fermentations without adding another costly enzyme. This information will be used by the dairy industry to produce new cost-effective health promoting products. A Material Transfer Agreement is under development with a commercial galacto-oligosaccharide producer.
Review Publications
Chaluvadi, S., Hotchkiss, A.T., Smith, B., Koerfer, B., White, A.K., Guron, G.P., Renye Jr, J.A., Yam, K.L. 2024. Key kombucha process parameters for optimal bioactive compounds and flavor quality. Fermentation. 10(12). https://doi.org/10.3390/fermentation10120605.
Guron, G.P., Hotchkiss, A.T., Bodnar, B.H., Harron, A.F., Renye Jr, J.A., McAnulty, M.J. 2025. Oligosaccharide production using ß-galactosidase from Lactobacillus bulgaricus and Kluyveromyces lactis in sweetened reconstituted nonfat dry milk. Journal of Dairy Science. 108(6):5696-5704. https://doi.org/10.3168/jds.2025-26396.
Muhidinov, Z.K., Nasriddinov, A.S., Strahan, G.D., Jonmurodov, A.S., Bobokalonov, J.T., Ashurov, A.I., Zumratov, A.H., Chau, H.K., Hotchkiss, A.T., Liu, L.S. 2024. Structural analyses of apricot pectin polysaccharides. International Journal of Biological Macromolecules. 279. https://doi.org/10.1016/j.ijbiomac.2024.135544.
Martorano, T.M., Ferguson, K.L., Cameron, R.G., Zhao, W., Hotchkiss, A.T., Chau, H.K., Dorado, C. Production of pectic oligosaccharides from citrus peel via steam explosion. Foods. 13(23):3738. 2024. https://doi.org/10.3390/foods13233738.
Mainali, K., Yadav, M.P., Sharma, B.K., Sarker, M.I., Lew, H.N., Hotchkiss, A.T., Simon, S. 2024. Isolation and characterization of the physiochemical properties of brewer’s spent grain. Agriculture. 15(1). https://doi.org/10.3390/agriculture15010047.