Metabolic and endocrine effects of a 12-week allulose-rich diet

This publication, in a special issue of 'Diabetes, Diet and Health Conditions', was published on June 10, 2024.

Source: MDPI

Authors: Kevin B. Cayabyab 1, Marley J. Shin 1, Micah S. Heimuli 1, Iris J. Kim 1, Dominic P. D'Agostino 2 ORCID, Richard J. Johnson 3, Andrew P. Koutnik 4, Nick Bellissimo 5, David M. Diamond 6 ORCID, Nicholas G. Norwitz 7, Juan A. Arroyo 1, Paul R. Reynolds 1, ORCID and Benjamin T. Bikman 1 *

1.) Department of Cell Biology and Physiology, Brigham Young University, Provo, UT 84602, USA

2.) Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL 33602, USA

3.) University of Colorado School of Medicine, Aurora, CO 80045, United States of America

4.) Sansum Diabetes Research Institute, Santa Barbara, CA 93105, USA

5.) Faculty of Nutrition, Toronto Metropolitan University, Toronto, ON M5S 1A8, Canada

6) Department of Psychology, University of South Florida, Tampa, FL 33602, USA

7.) Harvard Medical School, Boston, MA 02115, United States of America

* The author to whom correspondence should be addressed.

Abstract

The global increase in type 2 diabetes (T2D) and obesity necessitates innovative dietary interventions.

This study showed allulose to reduce blood sugar levels in a diet-induced obesity-induced T2D rat model.

Over 12 weeks, we hypothesized that allulose supplementation would improve body weight, insulin sensitivity, and glycemic control.

Our results showed that allulose alleviated the adverse effects of high-fat and high-sugar diets, including reduced weight gain and improved insulin resistance.

The allulose group showed lower food intake and increased levels of glucagon-like peptide-1 (GLP-1), enhancing glucose control and appetite regulation.

Additionally, allulose prevented the accumulation of hepatic triglycerides and promoted mitochondrial uncoupling in adipose tissue.

These results suggest that allulose supplementation may improve metabolic health markers, making it a promising dietary ingredient in the treatment of obesity and T2D .

Further research is needed to explore the long-term benefits and mechanisms of allulose in preventing and treating metabolic diseases.

This study supports the potential of allulose as a safe and effective intervention to improve metabolic health in the context of dietary overconsumption.

Keywords: insulin resistance; diabetes; obesity; mitochondria; allulose

1. Introduction

The increasing global incidence of type 2 diabetes (T2D) and obesity represents a significant public health challenge, necessitating the exploration of innovative dietary strategies to alleviate these diseases.

The rampant epidemic of type 2 diabetes (T2D) and obesity worldwide requires a relentless search for innovative and effective interventions.

Allulose (d-psicose) is a rare natural sugar that is found naturally in small amounts in certain fruits.

Allulose offers the sweetness of fructose, yet is metabolically different; it is the C-3 epimer of fructose, which, unlike fructose , has no effect on glucose or insulin.

Furthermore, unlike fructose, allulose is not a substrate for de novo lipogenesis and does not exert an inhibitory effect on fatty acid oxidation [ 1 ].

Taken together, these reasons make allulose an attractive candidate for dietary treatment of metabolic disorders, both by displacing dietary fructose and by providing putative metabolic benefits [ 1 , 2 , 3 ].

Unlike traditional sugars, allulose is largely absorbed in the small intestine and excreted without being fully metabolized [ 4 ], making it a low-calorie alternative to sucrose and high-fructose corn syrup, among other low- or zero-calorie sweeteners [ 5 ].

Recent human studies have highlighted the potential benefits of allulose, including improved glycemic control and reduced obesity, without the adverse metabolic effects associated with traditional sugars [ 1 , 6 , 7 , 8 ].

However, although it clearly enhances the release of endogenous glucagon-like peptide-1 (GLP-1) [ 9 ], which is widely exploited in weight loss drugs [ 10 , 11 ], the mechanisms underlying these beneficial effects remain to be fully elucidated.

Furthermore, while human studies are critical, animal models, particularly rodent models of type 2 diabetes and obesity, offer a valuable opportunity to examine physiological, metabolic, and molecular responses to allulose in a controlled environment.

This 12-week study examined the effects of dietary supplementation with allulose in a rat model of diet-induced obesity and type 2 diabetes.

We hypothesized that allulose supplementation would result in significant improvements in body weight, insulin sensitivity, and glycemic control compared to control animals fed a standard, allulose-free diet.

Additionally, we examined the potential mechanisms by which allulose exerts its effects, including its effects on adipocyte metabolism and adipokine production, hepatic nutrient management, and inflammatory markers.

By exploring the metabolic effects of allulose in a well-established animal model of type 2 diabetes and obesity, this study contributes to the growing body of literature supporting the use of allulose as a safe and effective dietary intervention for the treatment of these conditions.

The research findings may have significant implications for dietary recommendations, food industry practices, and the development of new therapeutic strategies targeting the etiology of type 2 diabetes and obesity.

The aim of this study was to confirm previous findings on the effect of allulose in alleviating some of the metabolic effects of a Western diet and to complement this by considering findings specific to adipose tissue physiology and mitochondrial bioenergetics.

2. Materials and methods

2.1. Animals

Twelve-week-old female and male Wistar rats were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and maintained at 22 ± 1°C on a 12-h light-dark cycle.

Animals were randomly divided into four groups and housed individually (n = 10; 5 females, 5 males) during a 12-week study as follows: standard laboratory chow with stevia (Stevita Naturals, Kennendale, TX, USA), Western diet chow with stevia, standard laboratory chow with allulose, or Western diet chow with allulose.

Allulose (allSWEET®) was provided by Anderson Advanced Ingredients (Irvine, CA, USA).

The animals had free access to food and water.

The Western diet was Research Diets D12266B (New Brunswick, NJ, USA), which contained sucrose, saturated fat (butter), and polyunsaturated fat (corn oil).

Stevia and allulose were administered in drinking water, and each animal received 30 ml of sterile water daily containing either 0.1 ml of stevia (sweetened to match the allulose) or 3% allulose (to achieve a daily dose of approximately 1.9 g/kg/day), as previously reported [ 12 ].

During the protocol, blood was collected from the tail vein every four weeks after an 8-hour fast for continuous hormone (insulin and GLP-1) and glucose analysis.

Following the 12-week protocol, animals were used for tolerance studies and then sacrificed for tissue sampling for further analysis (see below).

All work was approved by the Brigham Young University Institutional Animal Care and Use Committee (IACUC; 23-1222) and was performed in accordance with the procedures and regulations outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

2.2. ATP quantification

ATP concentration (n = 8 in each group) was measured using the ATPlite Luminescence Assay kit (Perkin Elmer; Waltham, MA, USA). Tissues were homogenized in ATP stabilization buffer (three volumes of ice-cold PBS containing 20 mM glycine, 50 mM MgSO4, and 4 mM EDTA).

The homogenates were then diluted 1:5 in ddH2O. A total of 100 μL of ATP stabilization buffer and 50 μL of mammalian cell lysis buffer were added to the wells used for standards in an opaque, white, 96-well plate and shaken for 5 min at 25 °C and 700 rpm on a shaker.

Then, 100 μL of the diluted samples were added to open wells, and 10 μL of the standards were added to the aforementioned ATP stabilization buffer.

The plate was then shaken for 5 min at 25 °C and 700 rpm on a shaker. 50 μL of substrate was then added to each well (in the dark) and shaken for 5 min at 25 °C and 700 rpm on a shaker. The solutions in the wells were left for dark adaptation for 10 min. Luminescence was then measured using a Victor Nivo Multimode Plate Reader (Perkin Elmer), and ATP concentration was normalized to protein concentration measured using a BCA protein assay (Thermo Fisher Scientific; Waltham, MA, USA).

2.3. Mitochondrial respiration

Mitochondrial oxygen consumption rates were determined at 37 °C from freshly isolated tissue using an Oroboros O2K Oxygraph (Oroboros, Innsbruck, Austria) with MiR05 respiration buffer, as previously described [ 13 ].

Before placing samples from adipose tissue into the chambers of the oxigraph respirometer, the samples were permeabilized in 0.05 mg/ml saponin (Sigma-Aldrich, St. Louise, MO, USA) in MiR05 for 30 min at 4 °C.

After addition of the permeabilized tissue, the respirometer chambers were hyperoxygenated to ~350 nmol/ml. Oxygen consumption rates were determined using the following protocol: Electron flow through complexes I and II was supported by the addition of glutamate + malate + succinate (10 mM, 2 mM, and 10 mM) to determine leaky oxygen consumption (GMS). After stabilization, adenosine diphosphate (ADP) (2.5 mM) was added to determine oxygen consumption associated with oxidative phosphorylation. After completion of the respiration protocol, samples were collected from the chambers and stored at -20 °C for further analysis.

Protein concentrations were measured using BCA assay (Perkin Elmer), and respiration rates were normalized to protein concentration.

2.4. Plasma protein analysis

Protein isolation was performed by homogenization with RIPA buffer, protease inhibitors (Fisher Scientific). Total protein was quantified using the BCA Protein Assay Kit (Fisher Scientific), and 20 µg of protein was used for active immunoblotting or protein characterization.

Samples were applied to membranes from custom murine antibody arrays (Abcam, Cambridge, UK) containing antibodies specific to C-reactive protein, active GLP-1, insulin, adiponectin and leptin and incubated overnight before being recovered and re-incubated with a second antibody array membrane.

Biotinylated antibodies were then added to each membrane and incubated overnight, followed by final incubation with a streptavidin-conjugated fluorescent marker to detect cytokine expression.

The membranes were imaged using the previously mentioned fluorescence imaging system (LI-COR; Lincoln, NE, USA) and then quantified using Image J version 1.54h (US National Institutes of Health, Bethesda, MD, USA).

Signal intensities were compared to positive controls on each membrane, as previously done [ 14 ].

2.5. Tolerance tests

The tolerance test was divided into three main procedures, which were performed on separate days, with at least a 48-hour recovery period between tests:

pyruvate tolerance test (PTT), glucose tolerance test (GTT) and insulin tolerance test (ITT).

For each test, blood samples were taken from the tail vein at 0 (baseline), 15, 30, 60, and 120 min after injection to measure blood glucose levels using a handheld glucometer (Bayer Contour; Whippany, NJ, USA).

After an 8-hour fast, rats were injected intraperitoneally with sodium pyruvate (2 g/kg body weight) to measure plasma pyruvate levels by enzymatic assay.

One week after PTT, GTT was performed after a similar fast followed by an intraperitoneal glucose injection (2 g/kg body weight).

ITT was performed one week after GTT, and rats were administered insulin (0.75 U/kg) intraperitoneally.

2.6. Liver examination

After euthanasia, liver tissue samples were collected, immediately weighed, and then homogenized in ice-cold buffer for determination of triglyceride and glycogen content.

Liver triglycerides were quantified using a triglyceride assay kit (ab65336; Abcam, Cambridge, UK) according to the manufacturer's protocol.

Glycogen levels were assessed using a liver glycogen assay kit (ab169558; Abcam).

Triglyceride and glycogen content were normalized to liver wet weight.

To assess liver function, plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured.

Blood samples were taken by cardiac puncture immediately after euthanasia, and plasma was obtained by centrifugation at 3000 g for 10 min.

Plasma ALT and AST activities were determined using commercially available assay kits (ab263883 and ab285264; Abcam).

2.7. Statistical analysis

Mean values ​​± SEM were evaluated using one-way (for single measurements) or two-way (for time-course measurements) ANOVA followed by Student's t-test.

The results are representative, and p-values ​​less than 0.05 were considered significant.

Statistical analysis was performed using GraphPad Prism 7.0 software.

3. Results

A central outcome of the study was to understand the effects of allulose on body weight changes and food intake, including the complementation of the mechanisms mediating such changes.

Body weight ( Fig. 1A ; male mean baseline weight: 328 ± 17 g; female mean baseline weight: 245 ± 12 g) was significantly increased in animals fed a Western diet (WD) containing stevia.

However, weight gain was significantly lower in the WD+allulose groups compared to the WD+stevia group.

It is important to note that no weight difference was observed between rats receiving stevia or allulose and those receiving the standard diet (SD), suggesting that the effects of allulose were specific to the Western diet.

Consistent with these results, food intake in the WD+stevia group was significantly increased compared to the SD+stevia group ( Fig. 1B ; male mean baseline intake: 21 ± 3 g; female mean baseline intake: 16 ± 2 g).

The WD+allulose group again showed a significant decrease in food intake compared to the WD+stevia group.

1. Figure 1. Body weight, food intake and endocrine changes during 12 weeks of allulose consumption.

During the 12-week study, the rats showed several significant changes.

First, allulose consumption reduced weight gain on a Western diet , which was reflected in a reduction in total food consumption.

Insulin (C) and glucose (D) levels, which were used to calculate HOMA-IR (E), were significantly increased in the WD+stevia group, while they were normal in the WD-+allulose group (n=8).

GLP-1 levels increased significantly in both allulose groups during the study ((F); n = 8). * p < 0.05; ** p < 0.005; *** 0.0005.

Consistent with the effects on body weight, allulose also blocked the development of hyperinsulinemia ( Fig. 1C ), hyperglycemia ( Fig. 1D ), and insulin resistance (based on the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) index; Fig. 1E ) observed in the WD+stevia group with the Western diet.

It is interesting to note that the protection provided by allulose was associated with higher serum levels of active glucagon-like peptide-1 (GLP-1; Figure 1F).

Of note, higher GLP-1 levels were observed in both allulose groups, but the benefit on body weight and metabolic parameters was only observed in the WD+allulose group.

To better understand the metabolic effects of allulose on whole-body metabolism, we performed tolerance tests, namely glucose, insulin, and pyruvate tests (Figure 2A–C).

In the case of intraperitoneal glucose load ( Figure 2A ), the WD+stevia group showed the largest glucose response, maintaining a significantly higher response throughout the test compared to the other groups (which is consistent with worse insulin resistance).

In contrast, the WD+allulose group elicited a moderate response.

Similar results were obtained with the insulin tolerance test (Figure 2B).

We also determined whether a Western diet could exacerbate gluconeogenesis by performing a pyruvate tolerance test.

As expected, the WD+stevia group showed significant stimulation of gluconeogenesis, with a pronounced increase in serum glucose levels due to pyruvate.

Of note, allulose blocked gluconeogenesis in rats fed a Western diet ( Figure 2C ).

Figure 2. Glucose, insulin, and pyruvate tolerance tests in allulose-fed rats.

At the end of the 12-week study, animals underwent tolerance tests for glucose (( A ); 2 g/kg body weight), insulin (( B ); 0.75 IU/kg body weight), and pyruvate (( C ); 0.75 IU/kg body weight) ( n = 6).

Based on the area under the curve (AUC), rats treated with WD+stevia showed the most dramatic response to all stimuli, with blunted responses observed in glucose ( A ) and insulin ( B ) tolerance tests for WD+allulose, while normal responses were observed to pyruvate ( C ). * p < 0.05; ** p < 0.005; *** p < 0.0005 vs. SD+stevia; # p < 0.05 vs. WD+stevia.

The primary tissue of interest for allulose is the liver and its differential nutrient processing, particularly glycogen and hepatic triglycerides.

Allulose has been shown to increase glycogen content in the liver of laboratory rats, resulting in some weight gain, despite having no effect on liver function or histology [15].

Consistent with this report, we also observed a significant increase in liver size and glycogen content in animals fed a standard diet with allulose compared to those fed stevia (Figure 3A,B).

It is known that the Western diet increases both glycogen and fat levels in the liver, and as expected, both glycogen and liver fat (triglycerides) levels were significantly elevated in the WD+stevia group (Figure 3A–C).

However, the WD+allulose group, although still showing high glycogen content, was largely protected from fatty liver development and developed less liver hypertrophy (weight gain) compared to the WD+stevia group ( Figure 3A–C ).

In fact, elevations in liver function tests were only observed in the WD+stevia group ( Figure 3E ).

These studies show that allulose has a protective effect on liver function outcomes of a Western diet.

In all groups, liver mitochondrial respiration remained unchanged ( Figure 3D ).

Figure 3. Characterization after 12 weeks of allulose consumption.

Liver mass (A) was measured before glycogen (B) and triglyceride (C) analysis after 12 weeks of Western diet (WD) and standard diet (SD) supplemented with stevia or allulose in drinking water.

Mitochondrial respiration (see Section 2) was measured to determine the direct effect of diet on liver function.

The liver enzymes alanine (ALT) and aspartate aminotransferase (AST) were quantified in plasma as markers of liver health (E). N = 8. *p<0.05; *p<0.005; *** p < 0.0005 vs. SD+stevia.

Due to changes in body weight, we sought to better understand the bioenergetic processes of adipose tissue.

We have previously found that various dietary interventions can increase the rate of fat metabolism [ 16 ]. Although in mitochondrial respiration ( Figure 4A ) or in ATP levels ( 4B Figure .) we did not observe any differences between the groups , we saw that the allulose groups had significantly lower respiration and ATP levels ( Figure 4C ), suggesting greater mitochondrial uncoupling.

Figure 4. Analysis of mitochondrial function of fat cells after 12 weeks of allulose consumption.

Mitochondrial respiration (( A ); see section 2 ) was measured in subcutaneous adipose tissue in rats after 12 weeks of Western diet (WD) and standard diet (SD) with stevia or allulose in the drinking water.

ATP was also determined (B) and a combination of variables was used to determine the extent to which tissues produced ATP based on respiration rate (C). N = 7. * p < 0.05 vs. SD+stevia.

Like the liver, the kidney also responds uniquely to allulose . After the 12-week study, we analyzed kidney weight (Figure 5A) and glycogen levels (Figure 5B).

Although there was no difference in kidney weight between groups, a slight but significant difference in kidney glycogen levels was observed in both allulose groups.

Figure 5. Kidney analysis after 12 weeks of allulose consumption.

Kidney weight (A) was measured before glycogen analysis (B) after 12 weeks of Western diet (WD) and standard diet (SD) with stevia or allulose in drinking water. N = 10. * p < 0.05 vs. SD+stevia.

To gain a deeper understanding of the impact of the intervention on the overall inflammatory and metabolic status, we measured the levels of several metabolic markers, including C-reactive protein (CRP), adiponectin, and leptin.

In animals in the WD+stevia group, CRP levels increased more than twofold compared to both SD groups ( Figure 6A ), this effect was blunted in the WD+allulose group.

Adiponectin levels were significantly reduced only in the WD+stevia group, while leptin generally followed a similar, albeit more modest, trend as in the case of CRP.

The adiponectin/leptin ratio, a proxy for adipocyte size [ 17 ], showed a significant decrease only in the WD+stevia group ( Figure 6B ).

Figure 6. Hormone levels in allulose-fed rats.

At the end of the 12-week study, plasma was collected from rodents fed a Western diet (WD) and a standard diet (SD) with stevia or allulose in their drinking water.

Analytes included C-reactive protein (CRP), adiponectin, and leptin (A). Adiponectin and leptin were also used to generate a ratio reflecting adipocyte size (B). N = 6. * p < 0.05; ** p < 0.005; *** p < 0.0005 vs. SD+stevia.

4. Evaluation

The results of this study contribute to the understanding of the metabolic effects of allulose, particularly in the context of diet-induced changes in body weight, food intake, insulin resistance, and tissue-specific metabolic responses.

Of note, our results support previous research findings that allulose supplementation in the Western diet alleviates the adverse effects often associated with high-fat and high-sugar diets, such as increased body weight and insulin resistance, which are of obvious importance worldwide [18].

Furthermore, this work provides new insights into GLP-1 dynamics as well as adipocyte changes and mitochondrial function.

Initial observations from our work support those found in previous work.

The observation that both the WD+stevia and WD+allulose groups significantly increased body weight compared to their counterparts following a standard diet (SD), albeit to different degrees, highlights the complex interaction between dietary sugars and weight regulation.

The lower weight gain in the WD+allulose group suggests a potential protective effect of allulose against diet-induced obesity .

This is supported by previous research showing that allulose has anti-obesity effects in both animal studies, primarily through reduction of visceral fat and improvement of lipid metabolism [5, 19, 20].

Furthermore, the moderate increase in food consumption observed in the WD+allulose group compared to the WD+stevia group may indicate an appetite-regulating effect of allulose.

This finding is consistent with studies suggesting that allulose may influence the satiety cascade and reduce total calorie intake [ 21 ].

The significant increase in active glucagon-like peptide-1 (GLP-1) levels in the allulose groups suggests another mechanism through which allulose may exert its metabolic benefits.

Levels of GLP-1, a hormone involved in glucose regulation and appetite control, have been shown to be increased by allulose intake, enhancing glucose tolerance and reducing weight gain [9, 22].

While others have shown increased GLP-1 secretion in response to allulose [23], we believe our results are the first to report increased GLP-1 levels in a given condition, suggesting that allulose exerts a sustained effect well beyond the point of consumption .

The differential responses of insulin and glucose levels between groups – where the WD+stevia group showed a gradual increase, while the WD+allulose group did not – further emphasizes the beneficial role of allulose in modulating glucose homeostasis and insulin sensitivity .

These data are consistent with the idea that allulose may improve glycemic control through both glucose storage and production, improving insulin sensitivity to enhance storage in muscle and reduce production from glycogenosis [1, 20], and positionally reducing enteral absorption through substrate competition [18, 24].

Our liver and adipose tissue analysis further illuminates the tissue-specific metabolic effects of allulose, including novel findings regarding changes in mitochondrial function.

While hepatic triglyceride levels were elevated only in the WD+stevia group, suggesting that allulose may prevent hepatic fat accumulation , the significant glycogen accumulation in all intervention groups, especially in the allulose groups, may reflect a shift in nutrient utilization or storage mechanisms.

This finding may partially explain the results observed by Liu et al. [ 15 ], who reported improved exercise capacity in rodents fed allulose.

Given that we and others have shown protection against obesity with allulose incorporation , we used our previous experience in analyzing mitochondrial bioenergetics processes in adipose tissue to determine the relevance of allulose in this process [13, 25].

We observed greater mitochondrial uncoupling in adipose tissue of both allulose groups, suggesting a novel role for allulose in this process. This mechanism may help explain the observed weight-regulating effects .

Due to tissue limitations, we were unable to directly measure uncoupling protein levels in adipose tissue to confirm changes in protein levels.

This study has some limitations. One limitation of the study is the use of stevia as a control.

We tried to use a control sweetener that is both common and mostly inert.

Because these products are so different, it is impossible to be sure that the doses of allulose and stevia used are “equal.”

Accordingly, we used doses of both that have been used previously [ 12 , 26 ].

It is important to note that while stevia has been shown to stimulate GLP-1 secretion in cell culture, we are not aware of any such evidence in rodents or humans.

This suggests that some of the benefits observed with allulose in this study may be specific to GLP-1 release.

Furthermore, stevia is an ideal control in this study, given previous evidence suggesting that stevia has no effect on weight gain indicators compared to water [27].

However, it should be noted the absence of a water-only group.

A second limitation is the lack of histological data for the liver and kidney.

Although our results suggest a beneficial effect of allulose supplementation on nutrient storage in the kidney and liver, and enzymes in the case of the liver, histological comparisons would have provided greater insight into the status of these tissues.

It is of utmost importance to search for food ingredients that can alleviate adverse metabolic conditions without compromising dietary satisfaction.

By providing new data and supporting previous work, our study demonstrates that allulose supplementation in the context of water deprivation can modulate body weight, food intake, and metabolic parameters in a manner that promotes improved metabolic health .

5. Conclusions

These findings highlight the potential for using allulose as a functional dietary supplement that can effectively improve metabolic health outcomes such as insulin resistance, body weight, and more, all without forcing potentially difficult dietary changes .

Further research is needed to explore the long-term consequences of allulose intake and its use in dietary strategies for the prevention and treatment of metabolic diseases.

Author contributions

Conceptualization, BTB, PRR, JAA, RJJ and KBC; methodology, KBC, MJS, MSH, IJK, DPD, APK, NB, DMD, NGN, PRR and BTB; software, KBC, MJS, MSH and IJK; formal analysis, KBC, PRR, RJJ and BTB; investigation, KBC, MJS, MSH, IJK, PRR and BTB; sources, PRR and BTB; data management, KBC and BTB; writing – preparation of the original draft, BTB; writing – review and editing, All authors; supervision, PRR and BTB; project administration, BTB; obtaining funding, BTB All authors have read and accepted the published version of the manuscript.

Institutional Review Board Statement

The animal experiment protocol was approved by the Brigham Young University Ethics Committee (23-1222; approved: December 13, 2023).

Data Availability Statement

Data available upon request.

Financing statement

This research was funded by Sponsored Research Agreement No. R0602714 awarded by Anderson Advanced Ingredients to Brigham Young University.

Footnotes

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