Author: Julia Dürr
Supervisor: Mohammed Hefni & Cornelia Witthöft Opponent: Björn Karlsson
Folate, choline, betaine, resistant starch & dietary
fiber in Swedish lentils: Effect of cultivar and growing
conditions
Degree project in Chemistry, 15hp
Abstract
Background: One key component in the development of sustainable farming and foodstuffs is to increase the cultivation of legumes, due to their environmental and health benefits. Legumes contain several essential vitamins and minerals, protein and fiber, but cultivation can be problematic due to their weak stem strength which results in loss of crops as a result of crop lodging and susceptibility to weed invasion. One possible solution is co-cultivation with cereal crops such as oats as they provide support and outcompete weed growth, however, it is still unknown if co-cultivation will affect the nutrient content of lentils.
Aim: The thesis has two aims: first, to compare choline, betaine, folate, resistant starch and dietary fiber content in two different types of lentils: Gotland lentils and Anicia lentils; and second to examine if co- cultivation with oats will affect the nutrient content of the two lentil types.
Methods: Betaine and choline were analyzed through high pressure liquid chromatography-mass spectrometry (HPLC-MS) and folate through high pressure liquid chromatography with fluorometric/UV detection (HPLC-FL/UV). Resistant starch, non-resistant starch and dietary fiber were analyzed according to enzymatic assay kits by Megazyme.
Results: Significant differences in nutrient content between Anicia and Gotland lentils were seen regarding resistant starch content, with Gotland showing a 50 % higher content. A significantly greater choline content was found in Anicia lentil samples that were co-cultivated with oats, showing approx. a 15 % higher choline content. Gotland lentils co-cultivated with oats showed a significantly greater choline, resistant starch and dietary fiber content by 15 %, 70 % and 10 %, respectively.
Conclusions: There was no reported significant difference in choline, betaine, folate and dietary fiber content, but in resistant starch between the two lentil types, with Gotland lentil showing a higher resistant starch content. There appeared to be a positive effect of co-cultivation with oats since a significantly higher choline content in both lentil types co-cultivated with oats and a significant increase resistant starch and dietary fiber in Gotland lentils co-cultivated with oats was reported. This suggests that co- cultivation can lead to an increase in nutritional content for some nutrients in Gotland and Anicia lentils.
The dietary fiber analysis confirmed that legumes are a great source of fiber by one portion providing approx. half the recommended daily amount. Cultivation and consumption of lentils can give both environmental and health benefits. Further studies are needed to explore other pulses and effect on other nutrients.
Sammanfattning
Bakgrund: En nyckelkomponent i utvecklingen av hållbara odlingsätt och livsmedel är att öka odlingen av linser på grund av dess miljö- och hälsofördelar. Linser innehåller ett flertal näringsämnen såsom vitaminer, mineral, protein och fibrer, dessvärre kan odlingsprocessen anses vara problematisk på grund av svag stamstyrka vilket kan leda till förlust av grödor eftersom de blir utsatta för invasion av ogräs.
En möjlig lösning för att underlätta odlingen av linser är att odla dem tillsammans med spannmålsgrödor som havre, vilket kan förse linsgrödorna med stöd och undvika invasion av ogräs. Det är okänt om odling av linser med spannmål påverkar näringsvärdet i linserna.
Syfte: Syftet var att jämföra mängden av kolin, betain, folat, resistent stärkelse och kostfibrer i två olika linssorter; Anicia och Gotlandlinser, och undersöka om samodling med havre påverkar näringsvärdet i dessa två linssorter.
Metod: Kolin och betain analyserades med high pressure liquid chromatography- mass spectrometry (HPLC-MS), och folat med high pressure liquid chromatography med fluorometrisk / UV-detektion (HPLC-FL / UV). Resistent stärkelse och kostfibrer analyserades med enzymkit från Megazyme.
Resultat: Signifikanta skillnader i näringsvärde mellan de två linssorterna Anicia och Gotland syntes i mängden resistenta stärkelse, där Gotlandlinser visade ett 50 % högre mängd. En signifikant ökning i kolinmängd syntes i Anicialinser som samodlades med havre, med en kolinmängd som var cirka 15 % högre. Gotlanlinser odlade i samband med havre visade även en signifikant ökning i kolin (15 %) resistent stärkelse (70 %) och kostfiber (10 %).
Sammanfattning: Det rapporterades inga signifikanta skillnader i kolin, betain, folat och fiber, men i resistent stärkelsemängd mellan de två linssorterna, där Gotlandlinserna visade en högre resistent stärkelsemängd. Det syntes en positiv effekt av samodlingen med havre då en signifikant ökningen i kolin visades i både linssorterna samodlade med havre och en signifikant ökning resistent stärkelsemängd och kostfibrer i Gotlandlinser samodlade med havre. Detta föreslår att odling av linser i samband med havre kan leda till en ökning i linserna nutritionsvärde för kolin och resistent stärkelse.
Analysen som gjordes på fibermängden i båda linssorterna visade att de är en mycket god källa till fibrer, då en 150 g portion av kokta linser täcker halva det rekommenderade dagsintaget av fibrer. Odling och konsumtion av linser ger även miljö- och hälsofördelar. Fler studier kan behövas för att undersöka odling i samband med havre på andra näringsämnen och även andra baljväxter.
Key words
Lentils, methyl donors, choline, betaine, folate, HPLC, LCMS, dietary fiber, resistant starch.
Acknowledgements
I would like to thank my supervisors Prof. Dr. Cornelia Witthöft and Assoc. Prof
Table of content
1. Introduction ________________________________________________________________________ 1 1.1 Legume cultivation and harvest _______________________________________________________ 1 1.2 Methyl-donors ______________________________________________________________________ 2 1.3 Analysis of methyl donors ____________________________________________________________ 4 1.3.1 Quantification and analysis of choline and betaine with HPLC-MS ____________________________ 4 1.3.2 Quantification and analysis of folate with HPLC __________________________________________ 5 1.4 Dietary fiber _______________________________________________________________________ 7 1.5 Resistant starch ____________________________________________________________________ 7 1.6 Aim of thesis _______________________________________________________________________ 7 2. Material and methods _________________________________________________________________ 8 2.1 Milling and preparation of samples ____________________________________________________ 8 2.2 Determination of water content _______________________________________________________ 8 2.3 Analysis of betaine and choline content _________________________________________________ 9 2.3.1 Water extraction of betaine and free choline _____________________________________________ 9 2.3.2 Acid hydrolysis for total choline content _________________________________________________ 9 2.3.3 HPLC-MS quantification of choline and betaine __________________________________________ 9 2.4 Analysis of folate content _____________________________________________________________ 9 2.4.1 Sample preparation _________________________________________________________________ 9 2.4.2 Conversion of 5-CHO-THF to THF ___________________________________________________ 10 2.4.3 HPLC quantification _______________________________________________________________ 11 2.5 Determination of dietary fiber _______________________________________________________ 11 2.6 Determination of resistant starch _____________________________________________________ 11 2.7 Calculation and statistical analysis ____________________________________________________ 12 2.7.1 Calculations and equations __________________________________________________________ 12 2.7.2 Statistical analysis _________________________________________________________________ 12 3. Results ____________________________________________________________________________ 12 3.1 Betaine, choline and folate content ____________________________________________________ 12 3.2 Dietary fiber and resistant starch content ______________________________________________ 13 4. Discussion _________________________________________________________________________ 14 4. 1 Methyl donors – choline, betaine and folate ____________________________________________ 14 4.2 Resistant starch and non-resistant starch ______________________________________________ 15 4.3 Dietary fiber ______________________________________________________________________ 15 4.4 Year of harvest ____________________________________________________________________ 16 5. Conclusion ________________________________________________________________________ 16 Annex 1 – Equations used in calculations ______________________________________________________ 21 Annex 2 – Raw data for water content determination _____________________________________________ 23 Annex 3 – Raw data for choline and betaine content in lentil samples ________________________________ 24 Annex 5 – Raw data for dietary fiber and resistant starch in lentil samples ____________________________ 29 Annex 6 – Statistical analysis with two way-ANOVA test __________________________________________ 32
Figures and Tables
Figure 1 – Different folate forms
Figure 2 – One carbon metabolism with folate as cofactor Figure 3– Milled lentil samples in ultracentrifuge mill
Figure 4 – Flow diagram for sample preparation prior to folate analysis Table 1 – Lentil samples used for analysis
Table 2 – Choline and betaine content (mg/100g dry matter) in lentil samples Table 3 – Folate (µg/100g dry matter) content in lentil samples
Table 4 – Mean dietary fiber, resistant starch and non-resistant starch content (g/100g dry matter) in lentil samples
Table 5 – Dry matter content of Anicia and Gotland lentil samples
Table 6 – Free choline content (mg/100g dry matter) after water extraction
Table 7 – Betaine content (mg/100g dry matter) in Anicia lentils 2018 after water extraction Table 8 – Content of total choline (free and bound) (mg/100g dry matter) in lentil samples Table 9 – Betaine content (mg/100g dry matter) in lentil samples
Table 10 – Folate content of lentil samples, prior to conversion of 5CHO-THF to THF presented as sum of folic acid with individual forms THF, 5-CH3-THF and 10-HCO-PGA in µg/100g dry matter
Table 11 – Folate content of lentil samples after conversion. Folate content is presented as sum of folic acid with individual forms THF with 5CHO-THF, 5-CH3-THF and 10-HCO-PGA in µg/100g dry matter
Table 12 – Ash, fiber and protein residue and total dietary fiber for lentil samples (g/100 g) presented in dry weight basis (DWB).
Table 13 – Control samples for resistant starch analysis (g/100g) presented in dry weight basis (DWB) Table 14 – Resistant starch (RS) content (g/100g dry matter) for lentil samples, presented in dry weight
basis (DWB)
Table 15 – Non- resistant starch (non-RS) content (g/100g dry matter) for lentil samples, presented in dry weight basis (DWB)
Table 16 – Statistical comparison of the effect of co-cultivation with oats and harvest year of the two lentil types Anicia and Gotland
Abbreviations
10-HCO-PGA: 10-formyl folic acid 5-CH3-THF: 5-methyltetrahydrofolate 5-CHO-THF: 5-formyltetrahydrofolate
5,10-CH+-H4 folate: 5,10- methenyltetrahydrofolate AMG: Amyloglucosidase
BHMT: Betaine homocysteine methyltransferase FBP: Folate binding proteins
FLD: Fluorescence detector
GOPOD: Glucose oxidase/peroxidase reagent
HILIC: Hydrophilic interaction liquid chromatography HPLC: High performance/pressure liquid chromatography
HPLC-MS: High pressure liquid chromatography-mass spectrometry NP: Normal phase
PC: Phosphatidylcholine PE: Phosphatidylethanolamine PGA: Folic acid
RDS: Rapidly digested starch RP: Reversed phase
RS: Resistant starch RSe: Rat serum
SAM: S-adenosylmethionine
SAX: Strong anion exchange solid phase extraction SCFA: Short chain fatty acids
SDS: Slowly digested starch TDF: Total dietary fiber THF: Tetrahydrofolate
1. Introduction
1.1 Legume cultivation and harvest
Increasing the production and consumption of legumes such as beans, peas and lentils is seen as a key component in the development of sustainable farming and foodstuffs. Legumes have a number of documented health benefits due to several nutrients that they consist of, such as protein, fiber, minerals and methyl-donors including folate, choline and in some cases, depending on cultivation conditions, betaine (1). Expanding legume production, which is currently only grown on less than 2 % of arable land in Europe, could lead to a number of benefits including the reduction in use of agrochemicals, environmental impacts caused by fossil fuels and provide locally produced legume-based foods that contain several essential vitamins and minerals, protein and fiber (2). An increased production and consumption of plant foods, such as lentils, would not only lead to a decreased climate impact by the food industry, but would also allow limited land resources to be used more efficiently to feed a larger population (3).
Currently, only 2 % of the Swedish arable land is used for cultivation of pulses (4), whereof the majority is field beans and peas that are used for animal feed. Since the Swedish consumption trend of ecological and vegetarian food has strongly increased the last few years (5,6), the interest in pulses, especially lentils, has increased as the demand for them as a foodstuff increases and also that harvests have been successful in the southern of Sweden (2). The low production of pulses in Sweden signifies a missed business opportunity for Swedish farmers as well as the potential environmental benefits that come with pulse cultivation, that could be made use of. Increasing ecological cultivation of pulses as foodstuff could lead to an improved profitability for ecological famers and is also beneficial to be used as break crops between cultivation of other crops such as cereals. This is due to the fixation of nitrogen in the air in symbiosis with soil bacteria, which minimizes the need for additional nitrogen fertilizer (7,8) and allows effectivization of the whole cultivation system (9).
Even though the cultivation of ecological lentils has been shown to carry several benefits previously named, their cultivation can be problematic due to their weak stem strength which results in loss of crops due to crop lodging and susceptibility to weed invasion. These problems could be resolved through the co-cultivation of lentils with cereal crops, such as oats. Cereal crops could provide both support to the lentils and outcompete possible weed growth. It is however unknown if this suggested co-cultivation will affect the nutrient content of lentils (9).
A similar study (10) on the co-cultivation of legumes with oats has previously been carried out, investigating crop yield and crop growth. The method, called cereal-legume intercropping, is said to allow complete utilization of plant growth factors such as light, water and nutrients, which played an important role in situations of limited water resources. However, both oat and legume crops developed slightly better when grown in monoculture as they did not have to compete for plant growth factors.
Intercropping led to a somewhat slower growth and lower crop yield. However, intercropping was still presented as a more beneficial method as the same crop yield obtained through pure stands, or monoculture, required 29 % more land. The aforementioned study however did not analyze the effect of intercropping on nutrient content (10), as this study intends to.
The following nutrients: choline, betaine, folate, resistant starch and dietary fiber, were chosen in the present study due to their health benefits, which is further described in the upcoming text. One example is fiber, that can prevent several chronic diseases such as diabetes and cardiovascular disease (11). It has
previously been seen that the choline, betaine and folate contents vary in beans in different cultivation conditions such as location (12, 13), but it is however unknown if co-cultivation of lentils with cereal crops such as oats, will affect the nutrient content. If the cultivation method were to affect the nutrient content of legumes, the results of this thesis project can be used as an aid to find the best cultivation method to maximize both nutrient content which can be beneficial for public health, but also bring forth new information on how to cultivate ecological lentils. Since legume cultivation also has environmental benefits as previously mentioned, they are a potential contender to serve as a sustainable staple foodstuff in the Swedish, if not a global diet. The two lentil types used in this thesis are Gotland lentil, which is a Swedish preserved species, and Anicia lentil, a French green lentil. Both lentil types were ecologically cultivated at the SITES research center Lönnstorp, SLU Alnarp (9).
1.2 Methyl-donors
Methyl groups are essential for several cellular functions such as DNA methylation, normal cell function, phosphatidylcholine synthesis and protein synthesis. They can be acquired through dietary methyl-donors, such as folate, betaine and choline that serve as source of labile methyl groups. A diet deficient in dietary methyl donors suggest associations with disorders in protein synthesis, fatty liver and muscle disturbances, as well as birth defects such neural tube defects (14).
Choline is a compound that is involved in a number of processes in the human body including synthesis of neurotransmitters, lipid transport, methyl-group donation and synthesis of phospholipids that are an essential component in cell membranes and are involved in cell-membrane signaling. Choline is also an important component in memory and brain development in fetuses and is suggested to decrease the risk of developing neural tube defects. It has therefore been officially recognized as an essential nutrient by the Institute of Medicine in 1998. It is acquired through two ways: diet and de novo synthesis. De novo synthesis occurs through the methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). PC is the predominant phospholipid in the majority of mammalian membranes. This synthesis does not meet daily requirements and is why dietary intake is necessary (15). Dietary sources of choline include milk, eggs and fish. The Institute of Medicine, Food and Nutrition Board has set the adequate intake of choline at 425 mg/day for women aged ≥19, 450 mg/day for pregnant women, 550 mg/ day for lactating women and 550 mg/day for men aged ≥19 (14). Choline is present in two different forms:
as bound, or lipid-soluble such as the form phosphatidylcholine, or in water-soluble form such as free choline. The majority of choline in foods is in bound form, between 45-60 % (16).
Betaine, known as trimethylglycine, is also a methyl-donor. It is obtained from diet or is generated from choline (17) through an irreversible oxidation reaction catalyzed by enzymes choline dehydrogenase and betaine aldehyde dehydrogenase (16). Betaine serves as a methyl-group donor in the betaine homocysteine methyltransferase (BHMT) pathway where methionine is generated from homocysteine via betaine homocysteine methyltransferase. Methionine can be used to produce S-adenosylmethionine (SAM), which is the major methyl donor in the cell and has several cellular functions as previously named. Betaine is also a major osmolyte, that regulates cell volume and stabilizes proteins.
Concentrations of betaine in crops has been shown to be influenced by the amount of stress experienced, where a higher stress factor can lead to an increased betaine content (18). Sources of betaine in foods include wheat germs, spinach, beets and wheat bran (14). There are no dietary recommendations for betaine (19).
Folate can be referred to as a class of water-soluble B-vitamins, due to its presence in many different interchangeable forms. Five forms of folate quantified in this thesis are found in figure 1 below.
Figure 1 Different folate forms including folic acid (PGA), 5-methyltetrahydrofolate (5-CH3-THF), methyltetrahydrofolate (H4 folate /THF), 5-formyltetrahydrofolate (5-CHO-THF) and 10-formyl folic acid (10-HCO-PGA)
In some foods, folate is present mainly as reduced and methylated in the form of 5- methyltetrahydrofolate (5-CH3-THF). Its synthetic form, folic acid is used in supplements (20). Folate is essential for normal cell function, growth and DNA replication (21). Folate serves as an essential cofactor in the one-carbon metabolism, which is involved in multiple physiological processes such as the synthesis of purines and thymidine, epigenetic maintenance through homocysteine remethylation and amino acid homeostasis of glycine, serine and methionine. Folate metabolism can be used as an umbrella term for the set of transformations involved in one-carbon metabolism, which can be seen in figure 2 below.
Figure 2 One carbon metabolism with folate as cofactor. Folic acid is reduced via dihydrofolate (DHF) to THF by dihydrofolate reductase (DHFR) before becoming active. In foods, folate appears most often in the form of 5-CH3-THF. Betaine donates a methyl group to homocysteine in the betaine-homocysteine methyltransferase pathway (BHMT) (20)
A lack of dietary folate intake is linked to an increased risk of neural tube defects in developing fetuses which can result in anencephaly, spina bifida or fetal loss. In adults, folate deficiency can lead to anemia, and can possibly impact cardiovascular health (22). Folate is found in dark leafy greens, citrus fruits, beans and lentils. Folic acid can be found in fortified foods such as breakfast cereal, flour and bread or in dietary supplements (23). According to the Swedish National Food Agency, the recommended daily amount for folate, is for adults and youth over 14 years is 300 µg, for fertile women 400 µg and pregnant and breastfeeding women 500 µg (24). Dry lentil flour has been shown to contain 170 – 220 µg folate /100 g (25) and dry lentils have been shown to contain 220 – 290 µg/100 g, which means that 100 g of lentils could provide a significant amount of the daily recommended allowance of folate (26).
1.3 Analysis of methyl donors
Quantification of methyl donors including betaine, choline and folate in this thesis was performed using liquid chromatography with mass spectrometry (HPLC-MS) or high-pressure liquid chromatography with fluorometric/UV detection (HPLC-FL/UV). In HPLC-MS, a HILIC column was used.
1.3.1 Quantification and analysis of choline and betaine with HPLC-MS
Quantification of choline and betaine can be performed by HPLC-MS (27,28), as done in this thesis. To briefly explain, chromatography is used to separate and identify components of a mixture based on characteristics such as size, total charge and binding capacity to the stationary phase. Interactions between the stationary phase, which is placed in a column and mobile phase allows effective separations of molecules due to different molecular characteristics such as affinity to the stationary phase. In liquid chromatography, the mobile phase is liquid. (29). Liquid chromatography can be subdivided into different modes including reverse phase (RP), normal-phase (NP) and hydrophilic interaction liquid chromatography (HILIC) (30). In reverse phase chromatography, a nonpolar stationary phase and a polar mobile phase is utilized. In normal phase chromatography, polar stationary phases are combined with a non-polar or moderately polar mobile phase (31). In cases with very polar compounds, HILIC is used for analysis of polar solutes that are difficult to retain in normal phase HPLC. These polar solutes are
separated through the use of a polar stationary phase with a mobile phase consisting of a polar solvent containing water as a minor constitute. The mobile phase often contains 60-97 % acetonitrile, and 3-40
% water. Advantages with HILIC is that polar compounds are able to be retained in the in the stationary phase, which are otherwise eluted too quickly in RPLC (32).
Mass spectrometry functions by converting analytes to a charged or ionized state which are then analyzed according to their mass to charge ratio (m/z) (33). A direct detection of molecules based on well-defined and easily understood physical property, this is, mass, is made (34). Coupling of mass spectrometry to liquid chromatography is desired due to the sensitive and highly specific nature of MS (33). HPLC-MS techniques have therefore become a crucial role in many studies as it offers better specificity of detection, lower matrix interferences and high detectability of molecules that are present in very low concentrations. It is a popular method used to identify and quantify many different compounds and metabolites in a single sample (35).
For methyl-donor choline, sample preparation prior to analysis through HPLC-MS through both water extraction and acid hydrolysis were used in this thesis. This is due to that choline can either be analyzed in free or bound form. During analysis of free choline, or plasma choline (36) samples are prepared through water extraction (37), as it is water-soluble (16). The total choline content, consisting of both free and bound choline requires sample preparation firstly through acid hydrolysis. This is due to the bound form of choline, that is lipid-soluble (16), is present as phosphatidylcholine (37). Hydrolysis of phospholipids is therefore required, before sample preparation through water extraction for the total choline content. Betaine is often analyzed solely through HPLC-MS with sample preparation through water extraction (19).
1.3.2 Quantification and analysis of folate with HPLC
Quantification of folate can be done through high performance/pressure liquid chromatography (HPLC) (19) using reverse phase columns such as C18 HPLC columns. It is a class of liquid chromatography where the mobile phase passes through columns under 10-400 atmospheric pressure, which results in efficient separation of many bioactive molecules (30). HPLC can be coupled with a fluorescence detector (FLD), that measures the emission of light of molecules after they have been excited to a higher energy wavelength. The source of light is usually a deuterium or xenon flash lamp. FLD are very sensitive and are often used for detection of substances in limited or low concentrations. Care should be taken choosing a suitable buffer as they can cause background fluorescence. Another detector that can be paired with HPLC is a UV-detector, which is common due many compounds of interest absorb light in the UV-region (190-600 nm). Certain types of UV-detectors can be set to operate at the absorbance maximum of the analyte, and to change wavelengths during a chromatographic run if different analytes have different absorbance maximums (38).
Folate, in its several interchangeable forms and derivates, often occur as polyglutamates (39). Most foods contain a variety of folate forms (40), that require sample preparation prior folate analysis though HPLC. Sample preparation consists of three steps: extraction, or liberation from the matrix, deconjugation and quantification of the resulting folate (39). During sample preparation, folates are at risk of exposure to oxidative degradation, especially in exposure to light, oxygen and heat, which also makes it one of the most vulnerable vitamins that are easily lost during food processing (40).
During extraction there are a few different sample preparation steps. The first step is to homogenize milled or grounded samples with a buffer. The buffer has a very important role as folate stabilization is necessary both before and during analysis. Therefore, a suitable buffer than provides stability contains
preservatives such as 2-mercaptoethanol (39) or ascorbic acid to prevent oxidative loss and protect from interconversion. Additional exclusion from other factors causing oxidative degeneration includes flushing samples with nitrogen and using low temperatures and light (40). After homogenization, the samples are boiled for thermal denaturation of folate-binding proteins (FBP) and enzymes that may catalyze folate degradation or interconversion (39). FBPs are proteins that are bound to folate to increase their stability (41).
The next step is enzymatic sample preparation, which involves converting polyglutamates to mono- or diglutamate. This requires the enzyme γ-glutamylcarboxypeptidase, also known as conjugase or folate hydrolase. Sources of this enzyme can be found in chicken pancreas, rat liver or rat kidney. Rat plasma is known to give monoglutamates as an end product and it often suitable for use in HPLC (39), as HPLC requires deconjugation to monoglutamates before quantification (40). Deconjugate preparations are not commercially available and require preparation before usage. Treatment with folate conjugase is paired with trienzyme extraction where enzymes α-amylase and protease are used to further free folate from proteins and polysaccharides (39). To purify samples, which is necessary before HPLC quantification, methods such as solid phase extraction with commercial disposable cartridges can be used (40). There are several types of cartridges such as strong anion exchange (SAX) cartridges that are used for purification (39).
Quantification of folate often occurs by HPLC with UV- or fluorescence detection. UV-absorbance detection can detect all forms of folate but does not have good sensitivity if samples are in very low concentrations. Fluorescence detection provides a greater level of sensitivity and specificity (39). Other methods of quantification include microbiological assays procedures with lactic acid bacteria.
Application of microorganisms to the quantitative determination of vitamins such as folate began when the realization that microorganisms require specific nutrient factors to grow occurred. The most commonly used microorganism for folate analysis is Lactobacillus rhamnosus due to its response to the widest variety of folate derivatives. Microbiological assays are done through 96-well microtiter plates, that are read with an automated plate reader using spectrophotometry (39).
One difficulty associated with HPLC analysis is that there is a lack of valid purification methods suitable for the food matrices. This results in hindrance for accurate analysis of individual folates. There are promising purification methods such as strong anion exchange, that however need to be tested and validated. HPLC quantification of folate requires good analytical skill and involves a complex extraction procedure. It has been shown in previous studies that HPLC quantification results in lower values, up 50% for the sum of folates compared total folate as quantified by microbiological assay. Why this can occur is believed to be due to the limitations of HPLC detectors to quantify some folate derivatives and complex sample preparation procedures, resulting in loss of the more sensitive compounds that may cause lower folate results (39). A lack of folate standards, and certified reference material also limitations in folate quantification (42).
One form of folate that has shown difficulties during quantification is 5-CHO-THF, due to weak fluorescence response (FLD) and ultraviolet (UV) absorption of the molecule, resulting in masking of the compounds peak. To improve the quantification of 5-CHO-THF, a method to convert 5-CHO-THF to THF has been developed by Hefni et al. (19). Through acidification of 5-CHO-THF with HCl, 5- CHO-THF is converted to 5,10- methenyltetrahydrofolate (5,10-CH+-H4 folate), and then reduced to THF by using sodium borohydride. It was seen that >95% of 5-CHO-THF was converted to THF, with
<5% being converted to 5,10-CH+-H4 folate. By adjusting the amount of HCl used, stability of all
individual folate forms was seen, meaning that this method can be used to obtain more accurate quantification of 5-CHO-THF (19).
1.4 Dietary fiber
The term dietary fiber is a broad one, defined by the Codex of Alimentarius Commission in 2009 as
“carbohydrate polymers with ten or more monomeric units, which are neither digested nor absorbed in the human small intestine” (43). Different classifications are used to differentiate between different sorts of dietary fiber such as origin, chemical composition and physiochemical properties such as fermentability, viscosity and solubility which can influence the therapeutic effects of consumption. A few different types of fiber include cellulose, pectin, inulin, resistant starch and polydextrose (44). Diets high in dietary fiber are linked to a number of health benefits such as fewer cases of cardiovascular disease, in gut health, appetite control and lower risk of type II diabetes. Foods high in fiber include whole grains, vegetables, fruits and legumes (45). Previous studies have shown that the dietary fiber content of lentils is around 16-21 g / 100 g dry matter (46,47). According to the Swedish National Food Agency, 100 g of cooked lentils contains around 9 g fiber (48), that can serve as a good source of fiber as many Swedes do not reach the recommended daily amount of 25-35 g (49). Dietary fiber is usually analyzed using enzymatic-gravimetric methods, where the food sample is treated with enzymes to mimic the small intestines digestive process. Precipitation and filtration are used to separate the non-digestible precipitate which includes total dietary fiber, protein and inorganic material. Protein and inorganic material are quantified in separate analysis to be subtracted from the weight to determine total dietary fiber (50).
1.5 Resistant starch
Starch, our main energy source, can be categorized into rapidly digestible, slowly digestible and resistant starch, which is related to factors such as rate and extent of digestion and nutritional content (51).
Rapidly digested starch (RDS) is quickly digested and absorbed in the small intestine, often leading to a quick elevation in blood glucose levels. Sources of RDS include freshly cooked starchy foods. Slowly digestible starch (SDS) is digested slowly throughout the small intestine, which leads to a slower and more sustained glucose release and is found in most raw cereals (52). Resistant starch (RS) is starch that escapes enzymatic hydrolysis in the small intestine and is fermented in the colon (53). Sources include grains, seeds, fruit such as bananas (52) and, importantly, legumes (54). How resistant the starch is, depends on the proportion of amylose to amylopectin. The two glucose polymers differ in susceptibility to enzymatic hydrolysis by amylase. Amylose is highly susceptible to enzymatic hydrolysis at the molecules branching points found at α 1-6 glycosidic bonds, while amylopectin is less susceptible due to its more linear structure with α 1-4 glycosidic bonds that are not easily hydrolyzed. Therefore, foods high in amylose are considered to be more ‘resistant’ to enzymatic hydrolysis. Other factors such as particle size and cooking processes can affect the digestibility of starches (55). Resistant starch has received attention due to its role in the production of short chain fatty acids (SCFA) during fermentation in the large intestine which has been associated with various beneficial health properties (53). Production of SCFA serves as an energy source and also participates in important immune modulatory roles (56).
Resistant starch content in legumes is shown to be around 5-7 % per 100 g dry matter (53,54). Resistant starch is often analyzed using enzymatic methods where the food sample is treated with enzymes such as amyloglucosidase and α-amylase to hydrolyze both resistant and non-resistant starch to glucose which is then measured with a glucose oxidase/peroxidase reagent (GOPOD) using spectrophotometry (57).
1.6 Aim of thesis
The aim of this thesis is twofold. Firstly, the study aims to compare folate, choline, betaine and dietary fiber content in two different types of lentils: Gotland lentils and Anicia lentils. Secondly, the thesis
aims to examine if co-cultivation with oats will affect the nutrient content of the two lentil types. The results on nutrient content will be used to examine how the different cultivation methods affect nutrient content of the lentils, but also to determine the most optimal way to achieve an increased nutritional content.
Therefore, the following thesis questions have been formulated:
o Does the content of choline, betaine, folate, resistant starch and dietary fiber differ between the two lentil types Gotland lentils and Anicia lentils?
o Furthermore, does the content of choline, betaine, folate, resistant starch and dietary fiber differ between cultivation in pure stands or co-cultivation with oats?
2. Material and methods
2.1 Milling and preparation of samples
The Gotland and Anicia lentil samples from Sveriges Lantbruksuniversitet were harvested year 2018 and 2019, and frozen before shipment prior to analysis in April 2020. Upon arrivement to Linnaeus University in Kalmar, the samples were milled using an ultracentrifuge mill (Ultra Centrifugal Mill ZM 200, Retsch; Fig 3). Samples were thereafter stored at -31°C prior to analysis.
Samples harvested in years 2018 and 2019 were taken from different sections of the cultivation area. For this reason, samples of the same lentil type and same treatment (with or without co-cultivation with oats), were combined to a composite sample each before analysis. The composite sample consisted of 3 g of each discrete sample, consisting of the same lentil type and cultivation method from different harvest locations of the same field. The samples that were used for analysis were then taken from the composite sample and named as according to Table 1.
Table 1: Lentil samples used for analysis.
YEAR OF HARVEST SAMPLE NAME
2018 Anicia 18
Anicia + oat 18 Gotland 18 Gotland + oat 18
2019 Anicia 19
Anicia + oat 19 Gotland 19 Gotland + oat 19 Samples co-cultivated with oats were marked “+ oat”
2.2 Determination of water content
Determination of water content in lentil samples was performed according the AOAC (58). In brief, duplicates of ~ 1 g of each sample were weighed and dried in metal cup holders at 105 °C overnight.
The samples were cooled in a desiccator for 1 hour and reweighed. The weight of the dried sample was subtracted from the total sample weight and multiplied by 100 to determine water content and dry matter percentage.
Figure 3: Milled lentil samples in ultracentrifuge mill
2.3 Analysis of betaine and choline content 2.3.1 Water extraction of betaine and free choline
The analysis of free choline content and betaine was based on the method by Hefni et al. (19). In brief, duplicates of ~ 0.1 g of each lentil sample, previously milled and frozen, was placed in a 2 mL centrifuge tube with 1.5 mL Milli-Q water, which were then vortexed for 5 minutes and centrifuged for 5 minutes at 13 000 x g. The supernatant was transferred to 15 mL Falcon tubes. The extraction was repeated once more, and the second supernatant was combined with the supernatant from the first extraction. 25 µl of combined supernatant was pipetted into a new Eppendorf tube. Ten µl of the internal standard mixture was added. The volume was adjusted to 1 mL using acetonitrile and methanol mixture (90:10). The supernatant was then filtered (Captiva syringe filters, Agilent) and transferred to labelled HPLC-vials.
2.3.2 Acid hydrolysis for total choline content
Analysis of the total choline content, consisting of both free and bound choline was based on the method Hefni et al. (19). Duplicates of 0.1 g of each lentil sample, previously milled and frozen, were homogenized through gentle stirring in 5 mL hydrochloric acid (1 M) in 100 mL Duran flasks and incubated at 60 °C in an oven for 18 hours. Thereafter, the samples were cooled to room temperature, the pH was adjusted to 5-6 using ammonium hydroxide, and the volume was adjusted to 10 mL using Milli-Q water. The samples transferred to glass tubes, were vortexed and centrifuged as 13 000 x g for 5 minutes. Two mL of the supernatant was transferred to a 2 mL centrifuge tube. Twenty-five µl of the supernatant was pipetted into an Eppendorf tube. 10 µl of the internal standard mixture was added. The volume was adjusted to 1 mL using acetonitrile and methanol mixture (90:10). The supernatant was then filtered (Captiva syringe filters, Agilent) and transferred to labelled HPLC-vials.
2.3.3 HPLC-MS quantification of choline and betaine
Choline and betaine were quantified according to the chromatographic conditions by Holm et al. (59) using LC (Agilent 1200, Agilent Technologies, Santa Clara, USA) coupled to a single quadrupole mass spectrometer (Agilent 6130). In brief, the chromatographic conditions consisted of a thermostated autosampler (5 °C), the mobile phase used was ammonium formate 25 mM: ACN (30:70), with a flow rate of 0.6 ml/minute. A HILIC column (ACE HILIC UHPLC/HPLC columns, Scotland) (3 µm, 150 mm x 4.6 mm) was used, with an injection volume of 20 µl and runtime of 20 minutes.
2.4 Analysis of folate content 2.4.1 Sample preparation
Sample preparation was performed in duplicate including trienzyme extraction (Fig. 2) and purification based on the method by Hefni et al. (60). Heat stable α-amylase (3000 U/mL) was obtained from Sigma Aldrich and used for sample extraction without any additional preparation. A protease suspension (5 mg/mL) was prepared by suspending 5 mg protease (Cat. No. P5147, Sigma Aldrich) in 1 mL 50 mM phosphate buffer with pH 6.1 containing 0.1 % 2,3-dimercapto-1-propanol (v/v). Rat serum (RSe) used for folate deconjugation as a source of γ-glutamyl hydrolase was prepared according to Patring et al.
(61).
Trienzyme extraction was performed with minor modifications according to Jastrebova et al. (62), where approximately 0.5-1 g of a milled sample was homogenised in phosphate buffer with heat stable α- amylase and then boiled in a water bath for extraction of folate (Fig. 4).The samples were then incubated at 37 °C with protease, for 1.5 hours, boiled to inactivate enzymes, cooled on ice and then centrifuged.
The supernatant obtained through centrifugation was adjusted to 20 mL with extraction buffer and incubated with heat stable α-amylase once more in combination with dialysed RSe for two hours at 37
°C for deconjugation. The samples were then once more boiled to inactivate enzymes, cooled on ice and centrifuged. Three and a half mL of the supernatant was purified with preconditioned SAX columns (HyperSep™SAX Cartridges, ThermoFisher) where the sample was eluded in 4 mL extraction buffer. Samples were thereafter placed in HPLC vials and flushed with nitrogen prior to analysis.
Figure 4: Flow diagram for sample preparation prior to folate analysis. Extraction buffer: 0.1 M phosphate buffer containing 2 % sodium ascorbate (w/v) and 0.1 % 2,3-dimercapto-1-propanol (v/v). Elution buffer: 0.1 M sodium acetate containing 10
% sodium chloride (w/v), 1 % ascorbic acid (w/v) and 0.1 % 2,3-dimercapto-1-propanol (v/v). SAX: strong anion exchange solid phase extraction. RSe: rat serum.
2.4.2 Conversion of 5-CHO-THF to THF
The conversion procedure of 5-CHO-THF to THF was performed according to Hefni et al. (19). A solution of sodium borohydride was prepared by solving 1.2 g NaBH4 in a small amount of elution buffer; 0.1 M sodium acetate containing 10 % sodium chloride (w/v), 1% ascorbic acid (w/v) and 0.1 % 2,3-dimercapto-1-propanol (v/v). The solution was transferred to a 100 mL volumetric flask and adjusted to 100 mL with elution buffer. For the conversion procedure, 1 mL of purified food extract or standard solution were placed in a 5 mL volumetric flask with 0.5 mL concentrated hydrochloric acid for 20 minutes for acidification. The volume was then adjusted to 5 mL with the NaBH4 elution buffer solution and left for 60 minutes to allow conversion. One mL of each sample was then transferred to HPLC vials and flushed with nitrogen prior to analysis.
The THF content of converted samples therefore contains not only THF but converted 5-CHO-THF.
To estimate the content of 5-CHO-THF, THF content in the non-converted sample was subtracted from the THF content in the converted sample and calculated as described in chapter 2.7.1 and annex 1 as by Hefni et al. (19).
2.4.3 HPLC quantification
Folate was quantified using HPLC (Agilent 1260, Agilent Technologies, USA), consisting of a quaternary gradient pump, thermostat autosampler (10 °C) and column compartment (23 °C), fluorescence detector with excitation and emission at 290/360 nm for THF and 5-CHO-THF, and 360/460 nm for 10-formyltetrahydrofolate (10-HCO-PteGlu) and a multiwavelength detector (280, 290 300 nm) for 10-HCO-PGA and PGA. The column used to separate folates was an ACE-column (3 µm, 150 x 4.6 mm) (Scantec Nordic, Sweden) under linear gradient elution conditions according to Hefni et al. (63) using phosphate buffer with pH 2.3 and acetonitrile with a flow rate of 0.4 ml/minute and injection volume of 20 µl with a run time of 42 minutes. Retention time was used to identify peaks.
Quantification was done through fluorescence detection and UV detection using a multilevel (n=8) external calibration according to Hefni et al. (63). For samples undergone the conversion procedure, an injection volume of 100 µl and run time of 35 minutes was used. Quantification of converted samples also using a multilevel (n=8) external calibration where calibration solutions were treated with the same conversion method as samples.
2.5 Determination of dietary fiber
The total dietary fiber (TDF) content was analyzed according to the AOAC method 985.29 (64) using the Megazyme Total Dietary Fiber Assay kit (K-TDFR-200A). In brief, the samples, together with heat stable α-amylase were cooked at 98-100 °C (30 min) for hydrolysis, depolymerization and gelatinization of starch. Incubation of samples with protease at 60 °C (30 min) follows to solubilize and hydrolyze proteins. A final incubation with amyloglucosidase at 60 °C (30 min) hydrolyses starch fragments to glucose. The samples were treated with four volumes of ethanol for precipitation of soluble fiber and removal of depolymerized protein and glucose from starch. Residues were filtered and washed with 78
% ethanol, 95 % ethanol and acetone, dried and weighed. Duplicates were analyzed for protein and ash content, to obtain the final weight of dietary fiber in the sample (65). Analysis of protein and ash content in dietary fiber samples for correction was performed according to AOAC methods (58).
2.6 Determination of resistant starch
Resistant starch content was determined according to the AOAC method 2002.02 (66) using the Megazyme Resistant Starch Assay kit (K-RSTAR). In brief, samples were incubated with pancreatic α- amylase and amyloglucosidase (AMG) in a shaking water bath at 37 °C for 16 hours for hydrolysis and solubilization of non-resistant starch to D-glucose. Upon addition of ethanol the reaction was ended, and resistant starch was recovered as a pellet through centrifugation. The pellet was then washed twice with aqueous ethanol (50%v/v) and centrifuged, and the supernatant was collected from all washings for non-resistant starch analysis. The pellet was dissolved in 2 M potassium hydroxide in an ice water bath during vigorous mixing with a magnetic stirrer, thereafter acetate buffer was added to neutralize the solution. AMG was added to hydrolyze resistant starch to D-glucose. A glucose oxidase/peroxidase reagent (GOPOD) was used to measure D-glucose in both non-resistant and resistant starch through spectrophotometry by measuring the absorbance at wavelength 510 nm against a reagent blank and a D- glucose standard (57).
2.7 Calculation and statistical analysis 2.7.1 Calculations and equations
The folate content is expressed as the sum of different folate forms in µg/100 g folic acid after conversion using their molecular weight. The equation is given in annex 1. Equations used for calculations of the dietary fiber and resistant starch content from the Megazyme assay kits (57,65) are seen in annex 1.
2.7.2 Statistical analysis
Data was presented as mean values (µg, mg or g/100 g dry matter) of duplicate samples (n=2). Statistical analysis was done using Excel to determine standard deviation (SD) and the coefficient of variation (CV). For quality control, the CV for means of duplicates were under 15-20 % for choline, folate, dietary fiber and resistant starch. For betaine, CV for means of duplicates were under 30 %. The program GraphPad Prism 8 was used for one-way ANOVA and Tukey’s multiple comparisons test with a level of significance set at α < 0.05 to compare nutrient content between lentil types and cultivation methods.
To compare if the cultivation method and harvest year had an effect on the different lentil types, an ordinary two-way ANOVA test and Tukey’s multiple comparisons test with a level of significance set at α < 0.05 was used.
3. Results
The water content analysis showed that the water content of the lentil samples was approximately 9 %, detailed data can be found in annex 2. The overall choline content of all lentil samples varied between 120-150 g mg/100 g dry matter, with the highest choline content in Gotland lentil + oat 19, and lowest in Gotland lentil 18. Free choline comprised ca 33 % of total choline, as shown for the Anicia lentil 18 sample (see Table 2 legend). Mean choline and betaine content are displayed in table 2. For betaine, the overall content in all lentil samples varied between 1.5 – 3.6 mg/100 g dry matter, with the highest betaine content in Gotland lentil + oat 19, and lowest in Anicia 18.
3.1 Betaine, choline and folate content
Table 2: Choline and betaine content (mg/100g dry matter) in lentil samples
Sample Harvest year Mean choline content
(mg/100g) Mean betaine content (mg/100g)
Anicia 2018 121.6B 1.8
2019 136.0 AB 2.0
Anicia + oat 2018 138.7AB 3.0
2019 145.9A 2.0
Gotland 2018 120.5B 1.5
2019 129.1 AB 1.5
Gotland + oat 2018 141.4AB –
2019 148.6A 3.6
Total choline and betaine content are expressed as mean values (n=2) with CV between 0-15% were accepted for choline, and 0-30% for betaine. CV above these values led to reanalysis of samples.
Free choline and betaine (after water extraction) in Anicia (mean, n=2) = 40 mg/100 g and 1.9 mg/100 g dry matter, respectively.
– = Faulty or contaminated sample unable to be quantified.
Different superscripts within the same lentil type represent significant differences (p<0.05).
No significant differences were found in betaine content within the lentil type.
For Anicia and Gotland lentil samples, the folate forms THF, 5-CH3-THF and 10-HCO-PGA were quantified. No peaks for PGA were identified. In samples that were not converted, no clear peak for 5-
CHO-THF could be identified.Therefore, 5-CHO-THF was indirectly quantified after conversion to THF and was ranging between 20-40 % of the sum of folic acid. The sum of individual folate derivatives was expressed as folic acid µg/ 100 g dry matter (Table 3), where the sum of folic acid for converted samples ranged between 170 – 260 µg/ 100 g dry matter. The highest folate content was found in Anicia lentils + oat 18.
Table 3: Folate (µg/100 g dry matter) content in lentil samples
Data are presented mean values (n=2).
THF: tetrahydrofolate, 5-CH3-THF: 5-methyltetrahydrofolate, 5-CHO-THF: 5-formyltetrahydrofolate, 10-HCO-PGA: 10- formyl folic acid. The sum of folate is expressed in folic acid µg /100 g as the mean sum of THF, 5-CH3-THF and 10-HCO- PGA (annex 1).
* = Quantified as THF after conversion of 5-HCO-THF to THF.
No significant differences were detected within the lentil types.
The variation between duplicate analyses <20 %.
3.2 Dietary fiber and resistant starch content
Table 4 includes mean values for dietary fiber, resistant starch and non-resistant starch. Dietary fiber varied between 16 – 120 g /100 g dry matter, with Gotland 18 containing the most dietary fiber, and Gotland + oat 19 containing the least. Mean resistant starch content varied between 4-7 g /100 g dry matter with the highest content found in Gotland + oat 19 and lowest in Anicia 18, Anicia 18 + oat and Gotland 18. Mean non-resistant starch varied between 30 – 40 g/100 g dry matter. Gotland + oat 18 showed the highest non-resistant starch content while Gotland + oat 19 showed the lowest non-resistant starch content.
Table 4: Mean dietary fiber, resistant starch and non-resistant starch content (g/100g dry matter) for lentil samples
Sample Harvest
year Mean dietary fiber
(g/100g) Mean resistant starch
(g/100g) Mean non-resistant starch (g/100g)
Anicia 2018 16.1B 4.1Bc 35.8
2019 19.8AB 5.1Bb 31.5
Anicia + oat 2018 16.3B 4.1B 32.5
2019 18.8AB 5.1B 38.0
Gotland 2018 18.9AB 4.1B,abc 37.9
2019 18.3A 6.6Aa 33.0
Gotland + oat 2018 17.9AB 5.3B 38.5
2019 16.7AB 7.0A 30.6
Dietary fiber is presented mean values (n=2).
Starch and non-resistant starch content are expressed as mean values (n=2) for all samples. Coefficient of variation (CV) between 0-15% was accepted
Sample
Harvest
year THF 5-CHO-THF* 5-CH3-THF 10-HCO-
PteGlu Sum as folic acid (µg/100g)
Anicia 2018 38.3 75.3 109.3 18.5 237.0
2019 41.8 54.8 57.0 13.2 165.2
Anicia + oat 2018 60.6 106.3 94.5 18.3 261.6
2019 47.0 46.2 60.6 16.2 168.4
Gotland 2018 63.0 79.2 93.3 11.8 245.1
2019 51.9 55.7 99.1 20.8 224.1
Gotland + oat 2018 39.9 95.2 90.1 17.4 239.0
2019 46.3 67.6 89.0 10.0 201.6
Different superscripts with capital letters within the same lentil type represent significant differences (p<0.05).
Different superscripts with minor keys between lentil types not co-cultivated with oats represent significant differences (p<0.05). No significant differences in non-resistant starch were detected within the lentil types.
4. Discussion
Overall, differences in nutritional content between the lentil types Anicia and Gotland lentils was only seen regarding resistant starch content, where Gotland lentils showed a 30-50% increase. However, a significantly greater choline content was found in Anicia and Gotland lentil samples that were co- cultivated with oats. Gotland lentils co-cultivated with oats also showed a significantly greater resistant starch content.
4. 1 Methyl donors – choline, betaine and folate
Regarding choline, Gotland and Anicia lentils co-cultivated with oats showed a significantly higher choline content of approx. 15 % compared to lentil samples not co-cultivated with oats. Since a portion of 150 g cooked lentils corresponds to 50 g dried lentils, Anicia and Gotland lentils not co-cultivated with oats would provide roughly 60 mg choline while Anicia and Gotland lentils co-cultivated with oats would provide roughly 70-75 mg, which is an increase of 5-10 %. This is however not of great practical importance in meeting the 6-fold higher recommended daily intake of choline (16). This also shows that lentils are not considered a rich source of choline.
According to the FoodData Central by the US Department of Agriculture (USDA) (67), the total choline content of 100 g raw lentils is 96.4 mg. Anicia and Gotland lentils showed a choline content between 120 – 150 g mg/100 g dry matter. The value for choline from the USDA’s database was calculated from different foods that were in the same food category as lentils and not lentils themselves, which could explain the discrepancy of data (64). Since no data is available on betaine and choline in the Swedish Food Composition database (48), more data from the Swedish context would thus be required to draw further conclusions on choline content in lentils.
To compare overall betaine content, to data from the USDAs database, the betaine content in all the lentil samples ranged between 1.5 – 3.6 mg/100 g dry matter. Similar foods such as raw soybeans, have been seen to contain 1.85 mg betaine /100 g (70), which can confirm the findings of betaine in lentils reported in this thesis.
It has been hypothesized that an increase in stress during cultivation can influence betaine content (18), leading to an increase and accumulation of betaine as it protects organisms against abiotic stress due to its role in osmoregulation (68). Co-cultivation with cereal crops such as oats provides might be a stress factor due to competition for growth resources such as light and water (10). However, no significant increase in betaine content was reported in the lentil samples co-cultivated with oats, which does not confirm such findings reported in previous studies (68). Other previous studies have however noticed a range in the ability to accumulate and even synthesize betaine in crops in response to environmental constraints (18,69), which may explain why no significant increase in betaine content was reported for varieties in this study.
The analytical variation between duplicates was high for betaine content, with one sample showing a CV of 46 % which can be due to the low concentrations of betaine in the samples. This may contribute to why no significant differences in betaine content between lentil types and cultivation method was
reported. Repetition of the analysis is needed to confirm the betaine content which was not possible due to the time limitations in this thesis.
The folate content of the lentil samples proved lentils to be a good folate source, ranging between 170 – 260 µg/ 100 g dry matter. Our data from Anicia and Gotland lentil samples confirm findings from previous studies showing 170 - 220 µg folate /100 g in lentil flour (15), especially since the samples were milled prior to analysis. The findings are also confirmed by previous studies reporting a folate content of 220 – 290 µg/100 g in dried lentils (26) which is also within the range of the folate content in Swedish lentils obtained through analysis. There was no significant higher content in folate in lentil samples co-cultivated with oats. However, the lentil samples can still be acknowledged as a rich source of folate, that can be of practical importance when increasing dietary folate intake. Since Riksmaten 2010 (66) observed that the intake of folate was below recommendations especially in young women who require a higher intake of folate (24), lentils could be a good candidate food to boost folate intake especially if the trend of vegetarian food is increasing (5,6).
To compare the folate content of the lentils with data from the Swedish Food Composition database, 100 g cooked lentils contain 73 µg folate (48), which means a 150 g portion of green lentils would provide approx. 20-30 %, or approx. 100 µg of the daily recommended intake, which can be seen as a substantial amount. Lentil samples analyzed in this thesis would provide approx. 80-130 µg folate per 150 g portion of cooked lentils, providing around the similar substantial amount of folate.
4.2 Resistant starch and non-resistant starch
The resistant starch content of all lentil samples ranged from 4-7 %, our data from Anicia and Gotland lentils. This confirms findings from previous studies reporting 5-7 % resistant starch content in lentils (53,54). The lentil type Gotland from the 2019 harvest showed a significant higher content in resistant starch compared to Anicia lentils from the 2018 and 2019 harvest, which suggests that lentil type may have an effect on some nutrient contents.
Gotland lentils co-cultivated with oats from the 2019 harvest appeared positively affected as they showed 70% more resistant starch compared to the Gotland lentils not co-cultivated with oats from the 2018 harvest. However, Gotland lentils not co-cultivated with oats in 2019 showed a significantly higher resistant starch content compared to Gotland lentils from 2018 and Anicia lentils both co-cultivated and not co-cultivated with oats, suggesting that the harvest year may have an effect on nutrient content.
There was no significant difference between non-resistant starch content in all lentil samples.
4.3 Dietary fiber
All lentil samples contained 16-20 g fiber /100 g, that confirms similar results in previous studies report a dietary fiber content of 16-21 g/100 g dry matter in legumes (46,47). The coefficient of variation for data on all lentil samples during the dietary fiber analysis resulted in 9 % (annex 5) which indicates that the analytical variation is low. Gotland lentils from 2019 showed a significant 20 % higher content compared to Anicia lentils from 2018 both with and without cultivation.
According to the Swedish National Food Agency’s database, one portion, 150 g, of cooked lentils are shown to contain 14.4 g fiber (48) which proves lentil as rich source of fiber considering that it will provide approx. half of the daily recommended intake of 25-35 g (49). Increasing the consumption of legumes could be beneficial as it means an increased consumption of dietary fiber. Only three out of ten people in Swedish population reach the daily recommendation for fiber, as observed in Riksmaten 2010
(71). These eating habits may however have changed since 2010, due to an increased awareness of fiber and its health benefits (11). The results obtained from analyses on Gotland and Anicia lentils can be considered a reminder of the rich fiber content of legumes.
4.4 Year of harvest
The lentil samples were collected from two different harvest years; 2018 and 2019 and cultivated with two different methods; co-cultivation and no co-cultivation with oats. To see if these two factors had an effect on the nutrient content in the two lentil types, a two-way ANOVA test was performed, where details are reported in annex 6. Anicia lentils appeared more affected by co-cultivation and harvest year than Gotland, showing mixed effects. Harvest year had a significant effect on the resistant starch content of the two lentil types, while co-cultivation had a significant effect on choline for the two lentil types.
Otherwise no shared trend was reported between the two lentil types by harvest year and the co- cultivation method.
5. Conclusion
With regards to the thesis questions, there was no reported difference in content of choline, betaine and folate but in resistant starch and dietary fiber between the two lentil types, with Gotland lentil showing an 50 % increase in resistant starch content and an 10 % in dietary fiber. There appeared to be a positive effect of co-cultivation with oats as a significant increase in choline content, resistant starch content and dietary fiber in lentils co-cultivated with oats was seen. This suggests that co-cultivation can lead to an increase in nutritional content for some nutrients in Gotland and Anicia lentils. The dietary fiber analysis confirmed that legumes are a great source of fiber by one portion providing approx. half the recommended daily amount.
The rich folate content in the lentil samples could be of practical importance by providing a substantial amount of dietary folate. Since there is at present state no data on the methyl donors choline and betaine in the Swedish National Food Agency’s database, this may highlight the importance of determining such values for better guidance in choosing foods with high betaine and choline content when aiming to improve dietary intake through consumption of Swedish pulses. However, this argument only applies to those who may choose to exclude animal foods from their diet as many animal foods are good sources of choline and betaine (69).
Co-cultivation of lentils may not only possibly lead to an increased nutrient content, as shown for lentils for the nutrients choline and resistant starch but even a better crop yield since less land would be required (10). Currently, only 3 % of pulses produced in Sweden end up on our plates, with the rest going to animal feed (72). Since there is an increasing demand for locally produced food and also vegetarian food (5,6), results from this thesis could therefore be a start in exploring the optimal cultivation method for lentils in regard to land area and nutrient content in Sweden, as cultivation in the southern parts of Sweden has already been successful (2). Further suggestion for future studies is to study other lentil types or even other pulses to examine if co-cultivation with cereal crops such as oats would improve the nutrient content and even to explore the ability to cultivate other pulses in Sweden to meet the increasing demand.
