L-proline Interaction with Methyl Linoleate Oxidation Products Formation in Dry System and at Temperatures: For Understanding in Low-Moisture Foods

Viral Shah; Gerald Buonopane; Louis Fleck

doi:doi:10.11648/j.ijnfs.20241302.15

Research Article |

| Peer-Reviewed

L-proline Interaction with Methyl Linoleate Oxidation Products Formation in Dry System and at Temperatures: For Understanding in Low-Moisture Foods

Viral Shah^*

, Gerald Buonopane

, Louis Fleck

Published in International Journal of Nutrition and Food Sciences (Volume 13, Issue 2)

Received: 20 March 2024 Accepted: 7 April 2024 Published: 29 April 2024

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Abstract

Dry and low-moisture foods could experience a significant loss in nutritional value due to the process of methyl linoleate oxidation. L-proline could interact with lipid oxidation products, potentially modifying their formation and reaction path. However, there was a lack of research on the interaction between L-proline and methyl linoleate oxidation products in dry and low-moisture food matrices, which was a concern given the potential impact on food safety and nutrition. To address this knowledge gap, a study investigated the interaction between L-proline and the oxidation products of methyl linoleate in a dry system. The study examined the formation of methyl linoleate oxidation products such as conjugated dienes, hydroperoxide, and hexanal in the absence and presence of varying moles of L-proline at different temperatures. The formation of conjugated diene, hydroperoxide, and hexanal was analyzed using UV spectrometer analysis, xylenol orange, and DNPH derivatization HPLC-UV analysis. The results showed that adding proline to methyl linoleate samples stabilized conjugated diene and decreased hydroperoxide and hexanal levels as temperature increased, compared to the control sample. This suggests that L-proline effectively interacted with methyl linoleate oxidation products and altered their formation and oxidation path in the dry system. Overall, this study provided a basis for significantly enhancing understanding of the reactions between L-proline and methyl linoleate oxidation products in dry and low-moisture foods, offered practical implications for the food industry, and paved the way for future research.

Published in	International Journal of Nutrition and Food Sciences (Volume 13, Issue 2)
DOI	10.11648/j.ijnfs.20241302.15
Page(s)	38-55
Creative Commons	This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.
Copyright	Copyright © The Author(s), 2024. Published by Science Publishing Group

Keywords

Oxidized Lipids, Amino Acid, Interaction, Co-oxidation, Arid System, High Oxidation, Autoxidation, Lipid Oxidation Markers

1. Introduction

Dry and low-moisture foods like edible nuts, seeds, meat, fish, and healthy food industrial products such as granola bars, cereals, and crackers are good sources of methyl linoleate (ML), providing numerous health benefits to consumers. However, their low water activity makes these foods more susceptible to ML oxidation than moisturized food

[1, 2]

. Therefore, it is crucial to understand how to provide consumers with the nutritional benefits of ML in dry and low-moisture foods

[3]

. The presence of L-proline amino acid can interact with ML oxidation product formation, reduce the amount of ML oxidation product, and alter the ML oxidation path in dry systems at various temperatures. Therefore, understanding the interaction between L-proline and ML oxidation products in the dry system is crucial before using it in the dry and low-moisture food matrix. By initiating the use of L-proline in ML containing dry food products, it could be possible to prevent the loss of ML nutritional benefits, which can help food industries and food scientists to develop new dry food products with the use of L-proline and ML for the benefit of consumers.

1.1. Lipid Oxidation in Food

Lipid oxidation is a complex process that occurs in food via autoxidation, photooxidation, enzymatic oxidation, ionizing radiation, and many other processes

[4, 5]

. Several factors influence this process, such as temperature, oxygen concentration, and prooxidants including transition metals, singlet oxygen, light, and temperature

[5]

. Food properties such as low moisture, intermediate moisture, fluid water region, water activity, surface area, emulsion, pH, antioxidant location, and solid fat content also play a significant role

[6, 7]

. Food scientists need to understand lipid oxidation in dry and low-moisture food. Specific food components such as amino acids, proteins, natural polyphenols, and metal chelators can exhibit antioxidant activity and inherent oxidative stability. While antioxidant systems can effectively mitigate oxidation, they may be disrupted during food processing, so the strategic placement of free radical scavengers within the food system is crucial. It is important to note that lipid oxidation can differ based on the location of the lipid within a food product

[8]

. Lipid oxidation products can react with several food constituents during processing and storage, reducing the nutritional value of the food items. These reactions can negatively impact the sensory qualities of the food. Therefore, our collective responsibility is to maintain the nutritional value and sensory quality of processed foods. Given the demand for such foods, understanding the complexities of lipid oxidation pathways in low-moisture foods is essential. ML is the lipid of choice for this study due to its unique properties. Its oxidation is more stable than that of methyl linolenic acid and oxidizes more readily than that of methyl oleic acid, making it an ideal candidate for our research. Furthermore, it has been extensively studied, providing a substantial base of existing data to compare and validate our results.

1.2. Introduction of Proline: The Unique Amino Acid

Proline is known for its ability to protect against reactive oxygen species, hydroxyl radicals, and free radicals. It prevents oxidation and damage to lipids, proteins, and DNA under high temperatures, UV exposure, and other oxidative stress conditions. Proline exhibits a protective mechanism that involves quenching singlet oxygen rapidly by forming a charge transfer complex, where it provides an electron. Proline reacts with hydroxyl radicals under hydrogen abstraction, forming the most stable radical. It carries the spin on the C-5 atom, which is distanced from the carboxyl group and proximate to nitrogen. This reaction rate with proline surpasses that of most other amino acids. During stress, plants accumulate high proline levels, which shield them from reactive oxygen species due to their singlet-oxygen quenching properties. L-proline can react with lipid radicals, alkoxyl radicals, hydroxyl radicals, and lipid secondary products, which might affect the formation of lipid oxidation products

[9-12, 14]

. The free L-proline amino acid is the choice for this study because other amino acids like histidine, tryptophan, lysine, and cysteine were studied in muscle meat, plant, animal, and dairy protein. In contrast, other components like carbohydrates, cellulose, enzymes, vitamins, and flavor were present, which can interfere with the study results of lipid oxidation products and amino acid interaction

[11]

. Our study on the role of free L-proline in the ML oxidation process in a dry system is unique and remains unexplored. Although research articles on the role of free L-proline in plants under stress conditions are available, we chose the free L-proline amino acid for our study as it presents a novel and uncharted research opportunity.

1.3. Significance of the Study

This study investigates the development of ML oxidation products in a dry system, both with and without L-proline. The dry system is designed to mimic a dry and low-moisture food environment. The experiment takes place outside the food matrix to eliminate interference from other food components and ensure better clarity of the results. The study better understands the interaction between L-proline and ML oxidation products, particularly in the context of the temperature effect and the dry system. The findings of this study have significant practical implications for the dry and low-moisture food-producing industry. Gaining insights into ML oxidation in L-proline presence can help design more effective formulations for dry food products and processing. The research analyzes the major ML oxidation products, which are theoretically and practically significant for understanding. Overall, this study promises to enhance our understanding of ML oxidation and pave the way for using L-proline in dry products containing ML.

The hypothesis is that radicals form during the oxidation of ML in dry food systems. These radicals interact with other compounds within the food matrix, stabilizing and altering the reactions. This process can substantially impact the formation of ML oxidation products. Moreover, the temperatures and hydrogen donor compounds influence the degree of this impact in a dry system. L-proline can alter the oxidation product formation and reaction path by donating hydrogen to radicals during oxidation and interacting with the products formed in ML oxidization. To rigorously test this hypothesis, measuring these products in varying amounts of L-proline at different temperatures is crucial.

This study aims to provide evidence of changes in the amount of ML oxidation products in the presence and absence of different amounts of L-proline under various temperature conditions in a dry system. The study will analyze conjugated dienes, hydroperoxide, and hexanal during the oxidation of ML in dry samples. The focus will be on short reaction times to detect reactions early in the oxidation process. The study investigates how different reaction temperature conditions (freezer, refrigerator, 25°C, 37°C, and 65°C) affect the distribution of ML oxidation products in the presence and absence of L-proline. The samples will be tested in a closed headspace system. The results obtained from the freezer and refrigerator temperature samples will provide information about products stored at lower temperatures, which can be compared with those stored at 25°C, 37°C, and 65°C. This comparison will help evaluate and understand the effects of elevated temperatures on the product. The study also examines the effect of different amounts of L-proline by preparing samples for control ML, mole ratio ML:L-proline::1:5, and ML:L-proline::1:10. A control sample of ML is used to obtain a result in the absence of L-proline, which is compared to the results obtained in the presence of L-proline. To investigate L-proline amounts, a sample with a higher amount of L-proline in ML (ML: L-proline mole ratio of 1:10) is prepared and compared to the results obtained from a sample with a mole ratio of 1:5. L-proline control samples will be prepared and stored with other samples at all five temperature conditions to avoid interference with the L-proline products. L-proline control will help evaluate whether any products will be generated from L-proline only in the absence of ML. The results of a control L-proline sample will be compared with ML and L-proline (1:5 and 1:10) to evaluate the accurate product formation of ML oxidation and L-proline.

2. Materials

Methyl linoleate was purchased from NU-CHEK PREP INC. L-Proline was purchased from Alfa Aesar. Isooctane was purchased from Fisher Chemicals. PeroxiDetect^TM kit (Catalog Number PD1), hexanal; 2,4-Dintrophenyl hydrazone of hexanal, 2,4-Dinitrophenyl hydrazine (DNPH), acetonitrile, dimethyl formamide (DMF), sulfuric acid and hydrochloric acid were purchased from Sigma Aldrich. All solutions were prepared using distilled water from the Milli-QTM water system.

Glass vials with cap, VWR; analytical balance, micropipette (200 µL, 100 µL, 2- 20 µL) and 1000 µL pipette, spectrophotometer (UV-5500 PC spectrophotometer Metash Program); freezer; refrigerator; oven; glass test tubes; Agilent HPLC 1260 Infinity II system (G711B Quat pump, G7129A 1260 vial sampler, G7116 1260 column compartment, UV detector), HPLC column Ultra C18 (150X 4.6 µm, 5 µm, cat# 9174565, Ser# 19052245), data acquire and analyze via Open LAB chromatography software, volumetric pipettes, volumetric glassware, centrifuge Allegra and HPLC vials.

3. Methods

3.1. Sample Preparation

For the control ML sample preparation, ML (8.9 mg) was weighed in a 5 ml glass vial and then capped. For the control L-proline sample preparation, L-proline (35 mg) was weighed in a 5 ml glass vial and then capped. For the ML:P:: 1:5 moles sample preparation, ML (8.9 mg) was weighed in a 5 ml glass vial, and L-proline (17.3 mg) was weighed in the same glass vial, which was then capped. ML and L-proline were mixed slowly with a vortex mixer. For the ML:P:: 1:10 moles sample preparation, ML (8.9 mg) was weighed in a 5 ml glass vial, and L-proline (35 mg) was weighed in the same glass vial, which was then capped. ML and L-proline were mixed slowly with a vortex mixer.

The samples were prepared in triplicate for each temperature condition and time point. The vials were placed in different temperature settings, including the freezer (-10°C), refrigerator (5°C), laboratory temperature (20°C), preset oven at 37°C, and preset oven at 65°C.

At each end of the day (days 1, 2, 3, 4, and 5), the sample vials were pulled out from each temperature and at time points. Then, triplicate vials were used for individual analysis for conjugate diene (using a UV spectrometer), hydroperoxide (using the PeroxiDetect^TM kit), and hexanal (using the HPLC-DNPH assay).

3.2. Conjugate Dienes Analyses

Conjugated dienes were analyzed using a modified AOCS Ti 1a-64

[13]

. 3 mL iso-octane was added into the sample vial and mixed well. ML in the mixed sample and control dissolved in iso-octane, while L-proline did not dissolve. So, all sample solutions were transferred into 5 mL centrifuge tubes and then centrifuged at 1430 RCF for 15 minutes to obtain a clear supernatant solution for analysis. A portion of the supernatant was retained for analysis, and the remainder was diluted serially by a factor of 50. The present sample solution of ML had a theoretical concentration of 200 nm/ml, while the serially diluted solution had a theoretical concentration of 4 nm/ml. The diluted sample solutions in iso-octane absorbance were collected from 200-300 nm and measured at 234 nm using a UV spectrophotometer

[15]

. The Iso-octane UV cut-off is 215 nm. The dilution factor was included in the calculation. The concentration was calculated using Beer’s Law with an extinction coefficient of 29,500 L/mol cm in iso-octane

[13, 20]

and converted in mmol/ mol of ML the following equations the following formulas (1), (2).

A=εlc(1)

\frac{mmol}{mol of ML} = \frac{\begin{matrix} Sample \\ absorabce \end{matrix}}{29500 \frac{L}{mol \times cm} \times cm} \times \frac{1000 X D}{\begin{matrix} mol of \\ ML \end{matrix}}

(2)

A= absorbance

ε. = the extinction coefficient 29500 L / mol cm

l = path length in cm

C = is the concentration in mol/L

1000 = to convert mol to millimole

D = dilution factors

3.3. Methyl Linoleate Hydroperoxide Analyses

The xylenol Orange Assay method is used to detect peroxide values, specifically lipid hydroperoxides, in organic, aqueous, and biological samples. The xylenol orange assay is highly sensitive and can detect nanomoles of ML hydroperoxides concentration in a sample solution. In this method, a reaction occurs between ferrous (Fe²⁺) ions and lipid hydroperoxide in the presence of acid, forming ferric (Fe³⁺) ions. When ferric ions react with xylenol orange in acidic media, a blue-purple complex (XO, 3,3’-bis [N, N-bis (carboxymethyl)aminomethyl]-o-cresol sulfone phthalein, sodium salt) is formed, which can be measured for its absorbance maximum between 560 nm. The reaction maximum occurs near pH 1.8, so sulfuric acid (H₂SO₄) is used in this assay for better sensitivity and precision. The tert-butyl hydroperoxide (t-BuOOH) is used for a standard calibration curve to calculate peroxide value

[16, 17]

. However, the extinction coefficient varies with the R group of the hydroperoxide, and the bleaching effect is a limitation of this analytical method

[18, 19]

Organic peroxide color reagent: The solution was prepared by mixing the contents of the vial (catalog number 08262 from PeroxiDetect^TM kit) with 120 mL of a 90 % methanol solution in water and mixing it well. After reconstitution, the solution contained 4 mM BHT and 0.125 µmole xylenol orange. Working Color Reagent for lipid hydroperoxides: 100 volumes of the reconstituted organic peroxide color reagents were mixed with one volume of Ferrous Ammonium sulfate reagent to prepare the resulting reagent solution containing 0.25 mM ferrous ammonium sulfate in 0.025 M sulfuric acid. 200 µM (0.2 mM) tert-butyl hydroperoxide (t-BuOOH) Standard Solution: To prepare a 1 M solution of t-BuOOH, a 70% (7 M) solution of t-BuOOH (Catalog Number 458139 from PeroxiDetect^TM kit) was diluted with methanol in a 7:1 ratio. For this, 0.5 mL of 7 M t-BuOOH was pipetted and transferred to a glass vial. Then, 3 mL of methanol was pipetted and transferred into the same glass vial. The total volume of the solution was 3.5 mL, and the vial was mixed well. Further, the 1 M t-BuOOH solution was diluted with three serial 10-fold dilutions in methanol to prepare a 1 mM solution. This 1 mM solution was then diluted 5-fold in methanol to prepare a 200 µM solution. To create the calibration curve, solutions for t-BuOOH, 0, 5, 10, 20, 40, 60, and 80 µL of the 200 µM t-BuOOH solution were pipetted and transferred to separate vials. The volume was adjusted to 100 µL using 90% methanol in water. Next, 1 mL of the working color reagent was added to each solution and mixed well. Then, the solutions were incubated at room temperature for 30 minutes to form the color. After that, the prepared standard solutions in the range of 1-16 nmoles of t-BuOOH per reaction volume were analyzed at 560 nm

[17]

. Table 1 presents the t-BuOOH linearity concentration range, correlation coefficient for the regression curve, slope, and intercept.

Table 1. The concentration range, the correlation coefficient for regression curve, slope, and intercept for the t-BuOOH calibration curve.

Name	t-BuOOH
Concentration range (nmol)	1 to 16
Intercept	- 0.095
Slope	0.0571
Coefficient of determination (R²)	0.9961
Regression equation	Y= 0.0571 X - 0.095

Lipid hydroperoxides, the primary oxidation products, were measured in triplicate using a xylenol orange commercial kit (PeroxiDetect^TM kit). Each sample was equilibrated to room temperature, followed by the addition of 200 µL of 90% methanol in water, vortexed briefly, and then centrifuged at 1430 RCF for 15 minutes to extract the hydroperoxide fraction. A volume of extract (80 µL) was removed and diluted to 100 µL using 90% methanol in water. Next, 1 mL of the working color reagent was added to each solution. The final mixture was mixed well and covered to prevent evaporation, and the solutions were allowed to incubate at room temperature for 30 minutes to form the color. Absorbance was measured at 560 nm on a spectrophotometer. Hydroperoxide concentration was calculated from a t-BuOOH calibration curve, and the results were converted in mmol/ mol of ML the following formulas (3).

\frac{\begin{matrix} peroxide \\ mmol \end{matrix}}{mol of ML} = \frac{(\begin{matrix} Sample \\ absobance - Blank \\ absorbance \end{matrix}) - Intercept}{Slope} X \frac{D}{10^{6} \times mol of ML}

(3)

Where,

10⁶ = conversation of nmole to mmol

3.4. Hexanal DNPH (2,4-Dintrophhenyl hydrazine) Assay Analyses

The aim here was to optimize reverse-phase high-pressure liquid chromatography (RP-HPLC) conditions and hydrazone solubility. The solubility of commercial C6-C10 DNPH carbonyls was evaluated in acetonitrile, acetonitrile: water:: 70:30, and dimethylformamide (DMF). Hydrazones were found to be soluble only in DMF. The DNPH hydrazone mixture was made up of saturated aldehyde C6-C10 and formaldehyde. Lipid oxidation produces various carbon chain-length carbonyl products. These could react with DNPH in the mixture and provide chromatographic interference at the hexanal-DNPH peak retention time. Therefore, it was necessary to separate other hydrazone peaks chromatographically from the hexanal-DNPH peak, which was crucial for accurate and precise analysis. DNPH-derivatized saturated aldehyde C6-C10 and formaldehyde were prepared under acidic conditions with DMF diluent, separated using acetonitrile-water gradients via RP-HPLC, and detected via UV. Literature-reported chromatographic conditions were used for sample analysis

[20]

. The RP-HPLC acquisition chromatographic conditions are presented in Table 2. The hydrazones eluted in sequence by carbon chain length. The shorter chain length eluted first, and the longer chain length eluted last.

DNPH acidic solution preparation: DNPH weighed 0.70 g and was transferred into a glass vial. DMF 9.9 mL was pipetted and transferred into the same vial. Mix well to dissolve. 0.100 ml Conc. HCl was pipetted and added to the DNPH solution, and the solution was mixed well. The DNPH concentration was 70 grams/little (357 mmole) in DMF. They were mixed well.

Table 2. Instrument condition for data acquisition.

Quaternary Pump (G7111B module), Autosampler (G7129A module), Column Compartment (G7116A module) and UV wavelength detector (G7114A module)
Flow (ml/min)	1.200 ml/min
Mobile phase A Channel	Water
Mobile phase B Channel	Acetonitrile
Gradient Timetable	Time (min)	A (%)	B (%)
	0.0	30	70
	15.00	0	100
	17.00	0	100
	18.50	30	70
	20.00	30	70
Stop Time (min)	20.00
Injection volume (µL)	10.00
Column	Ultra C18 5 µm 150 X 4.6 mm, Cat # 9174565
Signal wavelength	360 nm

The blank chromatogram shows no chromatography interference at the retention time for the peak of interest. The hexanal DNPH peak retention time was 7.06 min in the chromatogram. Table 3 presents the hexanal linearity concentration range, correlation coefficient for regression curve, slope, and intercept.

Table 3. The concentration range, the correlation coefficient for the regression curve, the slope, and the intercept for the hexanal calibration curve.

Name	Hexanal
Concentration range (µmol)	14 to 91
Intercept	31.77
Slope	22979.60
Coefficient of determination (R²)	0.9929
Regression equation	Y= 22979.60 X + 31.77

200 µL DNPH acidic was added to each sample vial and mixed well. They were kept in the dark for 30 minutes. Then, each sample solution was transferred to a separate microcentrifuge tube and centrifuged at 17530 RCF for 15 minutes. The supernatant solution was transferred to HPLC vials and analyzed via HPLC with detection at 360 nm wavelength

[20]

. The present ML sample solution had a theoretical concentration of 150 µmol/ml. A standard curve of µmol of hexanal was generated and used to calculate the hexanal results and converted the following formulas in mmol/ mol of ML (4).

\frac{mmol}{mol of ML} = \frac{(\begin{matrix} peak area \\ in sample \end{matrix}) - Intercept}{Slope} X \frac{D}{1000 X mol of ML}

(4)

Where,

1000 = to convert µmol to mmol.

D = dilution factor.

3.5. Statistical Analysis

All sample analysis experiments were conducted in triplicate, and the mean was compared using variance analysis. A significance level of p < 0.05 was defined as being statistically different. All calculations were performed using XLSTAT Excel data analysis software (version 2020.1.3, Addinsoft, New York, NY).

4. Results

4.1. Conjugate Diene Analyses

4.1.1. Conjugated Diene of Samples at Freezer and Refrigerator Temperature

The ML control and L-proline added ML samples (1:5 and 1:10) exhibited identical UV spectra at 234 nm when stored at freezer and refrigerator temperature conditions. There was no change in the absorbance at 234 nm wavelength for five days in ML control, L-proline control, and samples containing L-proline with ML from initial to five days. The concentration of conjugated dienes (mmol/mol ML) was calculated for the samples of ML control and L-proline added in ML (1:5 and 1:10) under freezer and refrigerator conditions with variable time points and results presented in Table 4 and Table 5. Results indicated the samples stored at freezer and refrigerator temperatures did not show a significant change in the formation of conjugated dienes in the presence or absence of L-proline in ML and support use of these temperature conditions samples served as temperature controls for the study of the room and elevated temperature conditions samples.

Table 4. Average conjugated dienes concentration (mmol/ mol ML) determined in samples stored at freezer temperature conditions.^*

Days	ML sample	ML:P::1:5 sample	ML:P::1:10 sample
0	5.15	6.97	6.98
1	6.05	6.15	6.16
2	6.32	6.00	7.44
3	5.76	5.89	7.41
4	6.43	6.55	7.36
5	6.87	6.03	6.50