Increasing natural

food folates through

bioprocessing and

biotechnology

Margaretha Jagerstada,*,Vieno Piironenb,

Caroline Walkerc, Gaspar Rosd,Emilia Carnovalee,

Marie Holasovaf andHeinz Naug

&

aDepartment of Food Science, Swedish University

of Agricultural Sciences, P.O. Box 7051,

SE 75007 Uppsala, Sweden

(Tel.: C46 18 671991; fax: C46 18 672995.;

e-mail: margaretha.jagerstad@lmv.slu.se)bDepartment of Applied Chemistry and

Microbiology, Viikki Food Science, University of

Helsinki, P.O. Box 27, 00014 Helsinki, FinlandcBrewing Research International, Lyttel Hall,

Nutfield, Surrey RH1 4HY, UKdFood Science and Human Nutrition, University of

Murcia, Campus de Espinardo, 300071 Murcia, SpaineInstituto Nazionale della Nutrizone (INN).

Via Ardeatina 546, 00178 Rome, ItalyfFood Research Institute Prague, Radiova 7, 102 31

Prague 10, Czech RepublicgDepartment of Toxicology, VMH Hannover,

Germany

The present study summarises results on processing effects

for folates obtained from an EU-funded folate project (QLK1-

1999-00576). Yeast fermentation, malting, germination and

Lactobacillus bacteria can be combined and further opti-

mised to potentially enhance the folate content in bread,

0924-2244/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.tifs.2005.03.005

* Corresponding author.

vegetables, dairy products and beer by 2–3 fold. Further

research, exploration and development of folate producing

lactic acid bacteria and yeast strains for food applications

should be encouraged. Milling technologies can be further

developed and by careful selection of raw materials and

ingredients, food processing can be designed and optimised

to increase folate content (and specific forms) using minimal

processing.

IntroductionCurrent data on folate intake in the EU population reveal

a gap between actual and recommended intakes. By

increasing the daily intake of folate in average between

100 and 200 mg, European consumers could benefit of

optimal function for health from folate, e.g. safer pregnan-

cies and maintenance of normal plasma homocysteine levels

and cognitive functions as well as protection towards certain

cancer forms, notably colon cancer (for ref, see Jagerstad,

Jastrebova, & Svensson, 2004). Limited bioavailability and

losses of folates during processing, storage, cooking, etc.

make the possibilities to reach recommended targets for

folate intake still rather uncertain. Fortification has been

proposed as one way to enhance folate intake. Another

option could be to develop foods including use of folate rich

raw materials and optimised food processing techniques to

enable the diet rich in folates within the range indicated to

be protective for human health.

Some food process techniques can enhance folate

concentrations, e.g. bioprocessing including fermentation.

Wine, beer and bread are examples of foods produced by

the fermentation of yeast, itself a rich source of folates

(Seyoum & Selhub, 1998). Lactic acid producing bacteria

(LAB) are other microorganisms used in food manufactur-

ing. LAB-fermented yogurt usually contains 2-fold higher

amounts of folate compared to the original milk, but as high

as 4-fold concentrations have been reported (Scott, 1989).

Bread baked from sourdough includes LAB and yeast

fermentation (Chavan & Kadam, 1989). Another example

is ‘idli’, steamed-fermented dough or mixture of rice

and black chick-peas consumed for breakfast in India.

Leuconostoc mesenteroides used as fermenting LAB for

‘idli’ production increased the folate concentration by

around 60% (Whitney & Ross, 1999). The increase in

folates in these products is explained by folate synthesis

from starter cultures.

Trends in Food Science & Technology 16 (2005) 298–306

Viewpoint

M. Jagerstad et al. / Trends in Food Science & Technology 16 (2005) 298–306 299

The objective of this work was to determine if, by careful

selection of raw materials and ingredients, food processing

can be designed and optimised to increase folate content

(and specific forms), and availability for absorption in key

foods using minimal processing and bioprocessing

(including yeast and fermentation microbes). Effects of

fermentation have been studied for cereal products, e.g.

bread and beer, milk products and vegetables. The stability

of added folic acid to flour during bread making of white

wheat bread was also investigated in order to gain knowl-

edge before a decision of an eventual folate fortification.

Material and methodsCereals (rye, wheat, oat and fortified white bread/rolls)

Ten rye varieties grown in Finland were obtained as

whole grains and milled at the laboratory. Grains, milling

fractions and rye products were obtained from Finnish

cereal companies. Samples of malted grains (rye, oats and

wheat) were obtained from processes carried out by Laihia

Malt Ltd., Finland.

Two approaches were applied to study the effect of

sourdough process on folates. First, the effect of different

baking methods was examined, and secondly, experiments

with yeasts and lactic acid bacteria were conducted at a

laboratory scale. Wheat and rye breads were baked using

different baking methods. Wheat breads were baked using

three methods: sponge-dough method, straight-dough

method and leavening with baking powder. Rye breads

were baked using three fermentations: leavened with yeast,

traditional sourdough fermentation with yeast and lactic acid

bacteria and sourdough fermentation with lactic acid bacteria

(without added yeast). In order to elucidate phenomena lying

behind the observed changes in folate contents during baking

process a study on folate contents and synthesis of different

yeasts and LAB was performed. Strains chosen to this

experiment are commonly present in sourdough or added as

starters. Microorganisms were grown both in a commercial

medium and in a mixture of rye flour and water.

Two types of breakfast roll (fortified with either high or

low folic acid content) were baked in different batches in a

commercial bakery (Skogaholm, Stockholm, Sweden). To

the first batch, 367.3 mg folic acid (Merck Eprova AG,

Schaffhausen, Switzerland) /100 kg dough was added to

give a folic acid concentration of 200 mg/roll. To the second

batch, 733.4 mg folic acid/100 kg dough was added to give

a concentration of 400 mg folic acid /roll.

BeersOne hundred and twenty commercial beer samples were

collected within EU. The beer samples differed in alcohol

concentration (0–10%). Typical beer varities included

Lambic beer (Meurice Institute, Belgium), Bavarian beers,

‘Weissbeer’ (Brewing school at Weihenstephan, Germany)

and Belgian trappist beers. Brewing process was studied

in pilot and commercial scale breweries covering the

three key steps: mashing, fermentation and packaging.

The commercial samples used to study the effect of

fermentation came from breweries in Germany. Pilot

brewing of a Bavarian Weissbiers was carried out in

collaboration with the German Brewing School at Weihen-

stephan. Two pilot samples of ale and lager were analysed

for folates before and after fermentation.

VegetablesThree different cultivars of beetroots (cv Boltardy, Ricky,

Kim) were grown in Southern Sweden and supplied by

Orkla Foods/Procordia Foods. Following washing, peeling,

and slicing, the beetroots were filled into glass jars together

with a water solution (2:1) containing acetic acid, sugar,

preservative (potassium sorbate) and spices. The filled jars

were locked by aluminium caps and pasteurised

(90 8C/40 min).

Coloured beans (cv Borlotto) produced in Italy in

July–August (fresh) and imported from North America

(dried) and corresponding canned beans (prepared from

fresh and dried seed) were supplied by Conserve Italia.

Domestic cooking of coloured beans was performed both on

fresh seeds (1 h cooking in salted tap water) and on dried

seeds (12 h soaking in tap water, 1 h cooking in salted

tap water). Two new varieties (Dinos and Globo) of green

peas were collected from Albacete (Spain). Fresh green

peas were washed, blanched (!85–90 8C/4–6 min), frozen

(K18 8C) and canned (110 8C/10 min).

Four fresh tomato varieties (cv Ronaldo, Tina, Cherry

‘rama’ and Cherry ‘pear’) were collected from Spain.

Samples were freeze dried before analysis. In addition some

other vegetables (red pepper, onion, cucumber, garlic) and

spices (laurel, parsley, oregano, dill) used as ingredients of

the tomato-based soup, Gaspacho were collected and

analysed for folates together with three brands of

pasteurised and sterilised Gaspachos, respectively and one

brand of tomato soup. Gaspacho ingredients were washed,

blanched, homogenised and pasteurised, into a final soup in

a factory and distributed aseptically packaged in Tetrabriks

(Alvalle). Pilot studies were conducted to optimise the

folate concentration by changes in formula, time of

homogenisation (1 0 vs 1.30 0) and particle size (thick; sieve

size 3 mm vs thin; sieve size!2 mm).

Two domestic samples of sauerkraut prepared from fresh

white cabbage were stored for 2 and 11 months, respecti-

vely, and analysed for folate with and without their juice.

Three commercial samples of canned sauerkraut (Dinapol,

Droga and Kuhne) were purchased from the local market.

Fermented vegetables were produced at Orkla Foods pilot

plant (Eslov, Sweden). Different vegetables (beetroots,

turnips, onion, white cabbage, carrot, pepper capsium

green, parsnip, celeriac) were washed, peeled, grated,

blanched (5 s in boiling water followed by draining)

re-mixed with apple juice containing 0.1% sodium salt

and inoculated with mixtures of different starter cultures

containing LAB, or propionibacteria (PAB) or bifidobac-

teria. The cultures were obtained from commercial culture

M. Jagerstad et al. / Trends in Food Science & Technology 16 (2005) 298–306300

producers (Christian Hansen, Danisco Cultor). The products

were packaged in plastic bags under vacuum. Fermentation

occurred in the dark for 4 or 7 days at 20 or 30 8C.

Unfermented samples were included as controls. Different

additional experiments were performed to optimise the

procedures before inoculation, e.g. varying the volume of

added water with and without additional ascorbic acid to

protect against folate oxidation and leakage during all

presteps. (For details, see Jagerstad et al., 2004).

FruitsFresh samples of yellow oranges (Italy), yellow grapefruit

(outside countries), pink grape fruit (Italy) and pineapple

(outside countries) were collected. Fresh red oranges (cv

Sanguinello, Moro, Tarocco) produced in Sicily were

provided by Parmalat (Italy). Two types of mild processes

were investigated for orange juices: a mild pasteurisation

industrial process and high-pressure system (600 Mpa/

4000 atm, ABB pilot plant). The mild pasteurisation process

was conducted by Parmalat and included squeezing of pooled

whole oranges, followed by freezing (K40 8C), pasteurisa-

tion (70 8C/15 s), chilling and packaging. The juice was

distributed and stored in refrigerator with a shelf-life of 40

days. The high-pressured process was conducted after

packaging at 600 Mpa, 5 min, room temperature with a

shelf-life of some months. In addition, high-pressured juice

produced at 400 and 600 MPa, respectively was conducted in

small-scale experiments by INRAN.

Milk and dairy foodsThirty commercial fermented milk products, 5 UHT

milks and 7 pasteurised milks were obtained from the retail

stores in Prague. Laboratory sterilised commercial UHT

milk or laboratory pasteurised raw milk was used. Selected

strains of Bifidobacterium longum, Bifidobacterium bifidum,

Streptococcus thermophilus and Propionibacterium

freudenreichii subsp. shermanii and ready to use butter

and yoghurt starters from the collection of dairy microor-

ganisms Lactoflora MILCOM Ltd (Czech Republic) were

used for model fermentation of milk. Laboratory sterilised

commercial UHT milk or laboratory pasteurised raw milk

was used in fermentations. This represented substrate with

minimal residual microflora and with relatively low content

of natural folate, allowing better recognition of changes

caused by fermentation. 5-Methyltetrahydrofolate analyses

were performed in time intervals (6, 12 and 18 h) during

fermentation process at 30 or 37 8C. In model samples the

individual strains as well as their combinations were tested.

Possible stimulants for folate syntheisis, e.g. glucose,

lactose, sodium ascorbate, inulin, cysteinHCl or p-amino-

benzoic acid (PABA) were studied.

In addition to folic acid fortification of white wheat bread

(see above) it was also found that milk could be a suitable

vehicle for folic acid fortification (Verwei et al., 2003). To

study the stability of added folic acid during processing,

95.5 mg folic acid (Eprova, Schaffhausen, Switzerland)

was added to 200 kg unprocessed milk. After fortification,

the milk was homogenised at 60–65 8C and divided into two

portions. One half of the milk was pasteurised (15 s, 76 8C)

and one half was processed following UHT treatment (15 s,

140 8C). Milk products were analysed for their total folate

content using microbiological assay.

Folate analysisSample preparation and folate analysis followed standard

procedures of published methods. All groups conducted

quality assurance tests on their methods including accuracy,

precision and repeatability. Certified reference materials

(CRM) obtained from the Institute for Reference Marterials

and Measurements (Geel, Belgium) were included in the

assays and all groups participated in a ring test during the first

year of the project to test proficiency of the methods. Food

samples were extracted by heating in the presence of

stabilisators (ascorbic acid, mercaptoethanol) followed by

deconjugation using hog kidney conjugase alone or in

combinations, so called trienzyme treatment (a-amylase,

protease and conjugase). Two ways of sample purification

were used prior to HPLC analysis: solid-phase extraction

(SPE) on strong-anion-exchange (SAX) isolute cartridges or

affinity chromatography. Total folate was analysed by

standard microbiological assay. Native reduced folate

forms were separated and quantified by HPLC following

the procedures described earlier (Jastrebova, Witthoft,

Grahn, Svensson, & Jagerstad, 2003; Kariluoto, Vahteristo,

& Piironen, 2001; Vahteristo, Lehikoinen, Ollilainen, &

Varo, 1997). The combined HPLC-microbiological assay

was used for certain samples. Standards of folate forms were

kindly provided by Merck Eprova and by Schircks

Laboratories, Switzerland.

Results and discussionCereals and cereal products

The folate content of rye grain is relatively high,

approximately 60–90 mg/100 g dry matter. 5-Methyltetra-

hydrofolate, 10-formyldihydrofolate and 5-formyltetra-

hydrofolate were shown to be the main vitamers.

However, both genetic factors and growing conditions

may cause some variation in folate contents. The coefficient

of variation for total folate contents among 10 varieties

grown in the same year was 8% (Kariluoto et al., 2001).

Furthermore, folates are concentrated to the outer layers of

the kernel and fractions taken from different parts of the

kernel can thus have significantly different folate contents.

Because milling of rye in technological scale can lead up to

40 different fractions, screening and adding folate rich

milling fractions to products offers a feasible way of natural

fortification. For example, Liukkonen et al. (2003) found as

large as 10-fold differences in the folate contents of the

inner kernel (appr. 10 mg/100 g dry matter) compared to that

in bran fraction (approx. 100 mg/100 g).

Germination is a biological process used in order to obtain

a typical flavour and texture. In germination, grain folates are

Fig. 1. Total folates in wheat and rye breads analysed by microbiological assay. Based on data in Kariluoto et al. (2004).

Table 1. Total folate content during rye bread process (backslopping) (based on data in Kariluoto et al., 2004)

Process step Folate content (mg/100 g dry matter)

Fermentation Aa Fermentation B

Rye flour 62 62Dough, back sloppingstart

71 58

Dough, back sloppingend

162 61

Dough after mixing 106 56Dough after leavening 111 54Rye bread 79 40

a S. cerevisiae added.

M. Jagerstad et al. / Trends in Food Science & Technology 16 (2005) 298–306 301

being synthesised as they are needed for growth and cell

differentiation. In industrially malted grains we measured the

folate contents to approx. 70 (oats), 140 (wheat) and 140–330

(rye) mg/100 g fresh weight. Malting of rye grains was used to

produce rye muesli with increased folate content, ca.

190 mg/100 g fresh weight. Liukkonen et al. (2003) also

showed significant folate synthesis; germination of rye grains

for 6 days was shown to lead to 1.7 to 3.5-fold increase in

folate content depending on germination temperature.

Germination more known as malting is also used in beer

brewing, resulting in a 2–3 fold increase in folate (see below).

For both wheat and rye breads the lowest folate contents

were found in breads baked without added yeast (wheat

bread leavened with baking powder and lactic acid bacteria

fermented rye bread) (Fig. 1). Wheat breads baked using a

sponge-dough or straight-dough method contained 2.5 times

more folates than bread leavened with baking powder. Also

in rye breads the difference was clear; lactic acid bacteria

fermented rye bread contained 31% less folates than bread

leavened with yeast or yeast and lactic acid bacteria

fermented bread. Inclusion of yeast resulted in higher folate

content of bread, in fact, the yeast addition resulted in

similar total folate concentrations in wheat bread and rye

bread, in spite of the lower folate content in wheat flour

(27 mg/100 g dry matter) compared to rye flour (44 mg/100 g

dry matter). Low amounts of tetrahydrofolate, 5-methylte-

trahydrofolate and 5-formyltetrahydrofolate characterised

breads baked without added yeast.

Another study was conducted to study the rye bread

process (Kariluoto et al., 2004). The total folate content in

sponge containing water, flour, baker’s yeast and a starter

culture with two lactic acid bacteria strains was significantly

higher at the end of the 16-h fermentation period (Table 1).

Dough making decreased the folate content due to the

dilution effect of adding other ingredients. Subsequent

proofing had little effect on total folate, whereas baking

resulted in some losses. Altogether, the folate content in

bread was 30–40% higher than in rye flour. However, folate

content did not increase during fermentation performed

without added yeast which further stresses the role of yeast as

the main contributor to folate content. Folate synthesis by the

yeast and different stabilities of the vitamers in acidic

conditions and later in baking led to changes in the vitamer

distribution. Increase in folate content during fermentation

resulted mainly from the increased amounts of 10-formyldi-

hydrofolate and 5-methyltetrahydrofolate. After baking,

practically no tetrahydrofolate was detected, and losses of

5-methyltetrahydrofolate and 5-formyltetrahydrofolate were

also significant.

Later in vitro fermentation experiments, conducted in a

mixture of rye flour and water, confirmed that fermentation of

Fig. 2. Summary of folate recovery during brewing—overall 20–30%relative to the starting ingredient, barley.

M. Jagerstad et al. / Trends in Food Science & Technology 16 (2005) 298–306302

rye has a clear influence on folate content and that microbes

differ in their ability to produce or consume folates. Yeasts

produced folates corresponding to 105–220% increase

whereas lactic acid bacteria isolated from sourdoughs did

not produce folates. Symbiotic relationship between yeasts

and lactic acid bacteria seemed to be possible although LAB

can retard the growth of yeast by competing for nutrients and

decreasing pH. Thus, factors affecting folate content in bread

include the microflora and amylolytic activity of rye flour,

starter cultures and baking conditions.

Folic acid fortified bread (rolls or loaves of wheat) was

used in some intervention studies. We aimed to produce

rolls with 200 mg folic acid/each. A 30% overage to adjust

for the losses during this test baking was used, but only

12% of the added folic acid were lost. In the final baking

the roll were produced with 400 mg (H) and 200 mg folic

acid (L), respectively. A 10% overage of folic acid was

added to compensate for losses during baking. However,

folic acid losses during baking were 25% in bread L and

19% in bread H. The folic acid concentration analysed by

HPLC was 166G47 mg in roll L and 355G63 mg/100 g

in roll H. Total folate concentrations in the rolls, ready

for consumption, quantified with the microbiological

assay were 198G80 mg/roll L and 365G145 mg/roll H

(Johansson, Witthoft, Bruce, & Jagerstad, 2002). For white

bread in forms of loaves the baking losses of fortified PGA

amounted to between 10 and 20%. Thus, if fortification of

wheat flour is introduced in Europe, these results indicate

that overages amounting to 10–25% are necessary.

BeersA survey of in total 120 EU beers was carried out, which

on average contained 80 mg folate/L ranging between 30

and 180 mg/L. In general, folate content was related to

alcohol content, which would be expected from the amount

of brewing raw materials used (higher alcohol required a

higher cereal content). Lambic beers were low in folate and

averaged 32 mg/L (nZ3). Also cheap beers were low in

folate as well as alcohol averaging 54 mg folate/L. Bavarian

wheat beers and Belgian trappist beers were both high in

alcohol and folate containing slightly above 100 mg

folate/L. Processing samples indicated that the high levels

may be due to the secondary fermentation step. For those

who consume beer regularly, folate intake from beer may be

in the order of 10–20% of the daily intake.

In order to optimise the retention of folate during brewing

various steps during processing were studied for the

recovery of folate (Fig. 2). Results are summarised as

follows:

The typical level of folate in unmalted cereals was

between 0.5 and 1.0 mg/kg, but during the malting process

the folate levels in barley increased 2 to 3-fold, particularly

during the first 2 days of germination. A survey of raw

materials showed that typical levels of folate in malt were ca.

2–3 mg/kg but special malts (crystal) and roasted products

had lower or negligible contents. Under pilot plant

conditions, folate losses during boiling and drub separation

range from 10 to 20%. Overall recovery of folate from the

raw materials for the whole process in commercial breweries

was approximately 25–30%.

For both lagers and ales, large folate losses were seen

during mashing and in-pack. Laboratory scale analyses

suggested that losses during mashing were a function of

time and temperature. In a commercial brewery, mashing

was also the key area for folate loss with losses ranging from

39 to 73% depending upon the type of beer, liquid:grist

ratio, mash regime and type of separator used. Initial results

suggest that shorter mashing times would favour increased

folate recovery from the malt. Optimising folate recovery

from mashing on the lab scale, it was found that a thinner

mash was beneficial. Also, avoiding prolonged exposure to

higher temperatures improved recovery. Other possible

parameters such as pH, fineness of the grind offered no

improvement in folate recovery. Optimisation of recovery

during mashing may be achievable by adjusting levels of

oxygen and mashing conditions in general (time/tempera-

ture) and this is an area for future work. Processes such as

de-alcoholisation and sterile filtration did not significantly

impact on folate content. Small losses were seen during

fermentation. Detailed studies showed folate levels to

increase during fermentation due to synthesis by the yeast

during the initial period of fermentation. However, this

folate was lost at the end of fermentation when the yeast was

removed indicating an intracellular localisation of yeast

folate. It was found that all yeast types tested behaved

similarly and no benefit could be found from adjusting

yeast type. Further investigation of folate vitamers using

HPLC-MA, demonstrated that 5-methyltetrahydrofolate

was the major folate form produced during fermentation.

Also 10-formyldihydrofolate increased, while folic acid and

5-formyltetrahydrofolate changed very little.

After an initial loss of folate during packaging, long-term

stability of folates was good, with little loss over 6 months

being recorded. Some beer styles, such as Bavarian wheat

beers, have a secondary fermentation, often in the bottle.

Pilot scale trials of ‘bottle-conditioning’ with varying yeast

M. Jagerstad et al. / Trends in Food Science & Technology 16 (2005) 298–306 303

concentrations suggested that this process increase folate.

In conclusion, although the levels of folate in beer are

significant, our data suggest that by careful selection of raw

materials (malt) and some changes in the brewing process,

beers with a higher folate content could be produced.

VegetablesMany vegetables, e.g. beans and green peas are

consumed after being cooked by boiling or re-heated from

canned products and it is well known that folate being a

water soluble B-vitamin, is easily lost by leakage. When

coloured beans were cooked domestically either from fresh

or dried beans, they ended up with similar folate

concentrations, around 25–30 mg/100 g. Starting amounts

of folate were 109 and 70 mg/100 g for dried and fresh

beans, respectively. Comparing the domestic cooking

figures with traditional canning (sterilisation) resulted in

similar folate concentrations (around 30 mg/100 g) for these

coloured beans (Table 2). As seen in Table 2, canning and

domestic cooking increased moisture dramatically. Calcu-

lations of folate retention based on dry matter showed

canning of beans to retain 72–75% of the folate while

domestic cooking of beans retained 5–10% more folate,

regardless if the beans were fresh or dried from start.

Canning of green peas after blanching and freezing

(55–65 mg total folate/100 g) resulted in 50% lower

amounts of folate. The other 50% was leaked into the liquid

Table 2. Total folate in raw and vegetables processed by pasteurisation

Vegetable N Moisture % Status

Beetrootsa 6 85 Raw, 3 varie3 82 Pickled/pas

Coloured beansb

(cv Borlotto)48 Fresh71 Canned, dra74 Domestic c16 Dried69 Canned and69 Domestic c

Grean peasc 6 73 Raw,6 75 Frozen6 77 Canned, dra

Tomatoesc 6 94 Raw3 72 Soupd

Gaspachosc

Commercial 6 94 PasteurisedCommercial 6 91 SterilisedLaboratory 6 89 Optimised

Vegetable mixturese Grated, blanVegetable mixturese Grated, blanWhite cabbagee RawSauerkraute 2 Fermented,

juiceCommercial sauerkraut 3 Soured and

a Grown in southern Sweden by farmers contracted by Orkla Foods.b Raw imported from North America, canned and supplied by Conserc Green peas: harvested 150 km NW Murcia (Spain), Tomatoes: harved Campbell’.e See Jagerstad et al. (2004).

(Table 2). Similarly, pickled sliced beetroots which were

preserved using milder processing, e.g. pasteurisation at

90 8C during 40 min, also resulted in around 50% folate left

in the beetroots—from 72–95 mg/100 g to 32–43 mg/100 g

(Table 2).

Another approach to enhance folate in processed

vegetables was performed by the Spanish group. The

tomato-based and popular soup, Gaspachos, was studied by

them. When comparing commercial brands that were either

sterilised or pasteurised none contained more than 5 mg folate

/100 g. Cambell’s canned pure tomato-soup contained 13 mg

folate per 100 g. Small-scale laboratory studies showed that

homogenisation time and particle size can be optimised and

reduce folate losses by residue. Another approach to increase

folate was by changing the recipe/formula, e.g. increasing the

amounts of folate-rich ingredients and add certain folate-rich

spices. Results of such trials showed increases of folate by

around 25% in Gaspachos. Overall, the folate losses

approached 85–90% in industrial processing owing to heat

degradation of folate and removal of incomplete homogen-

ised raw material containing folate. The laboratory-scaled

studies also revealed that preparing the Gaspachos instantly

from fresh vegetables resulted in folate concentrations

around 20 mg/100 g (Table 2).

The idea to enhance folate by changing the recipe/

formula initiated an extensive screening of various veg-

etables and spices. The objective of these studies was also

, sterilisation (canning), domestic cooking or fermentation

Analysed folate(mg/100 g FW)

Assay

ties 72-95 HPLCteurised, 3 varieties 32–43 HPLC

69 MAined 28 MA

ooking 29 MA109 MA

drained 26 MAooking 32 MA

HPLC59/70 HPLC/MA

ined 29/31 HPLC/MA7–16 HPLC13 HPLC

0.5–5 HPLC2–3 HPLC5–22 HPLC

ched and 15–16 HPLCched and fermented 10–17 HPLC

10–11 HPLCdomestically, including 18–20

canned, including juice 5–21 HPLC

ve Italia.sted 50 km E of Murcia (Spain).

M. Jagerstad et al. / Trends in Food Science & Technology 16 (2005) 298–306304

to update Spanish food tables with folate values based on a

validated HPLC method.

The possibility that folate concentrations could be

optimised by selecting the right cultivar/variety of veg-

etables was studied for tomatoes, green peas and beetroots.

Usually, the variation between different varities/cultivars

did not exceed 30%, with exception for one tomato cultivar

that contained less than half the concentration of folate

compared with the other three. Further optimisation of folate

in vegetables can be sought in improved harvesting

procedures and storage conditions.

Sauerkraut is a traditional fermented vegetable, produced

by spontaneous fermentation. Analysis of small-scale

products showed folate concentrations around

20 mg/100 g, while commercial products of Sauerkraut

manufactured by souring or fermentation followed by

canning contained between 5 and 21 mg/100 g (Table 2).

In collaboration with a Swedish food company (Orkla

Foods) commercial starter cultures aimed for manufacture

of fermented dairy products were subjected to mixtures of

grated and blanched root vegetables, mainly beetroots and

turnips. After fermentation periods between a couple of

days and 2 weeks either in room temperature or at 30 8C,

the results indicated that among 10 different LAB

cultures, one mixture was superior, resulting in almost a

doubling of folate concentration, mainly as 5-methylte-

trahydrofolic acid. The increased folate concentration was

seen when the figures were corrected for leakage. Leakage

of 25–50% of the folate took place during the overall

process, since water was added and removed at different

steps (cutting, blanching, and fermentation). Thus, the

overall retention of folate ended up in 50–75% when

calculated on wet weight. And the concentration of the

folate was similar in the surrounding liquid medium as in

the solids of the final fermented product. Interestingly,

one species of propionibacteria produced vitamin B12

(Jagerstad et al., 2004).

FruitsTropical fruits belong to the most folate-rich ones and

were studied by the Italian group. Fresh red oranges

contained the highest amounts of folate, between 45 and

50 mg/100 g, followed by the yellow oranges

(40–45 mg/100 g). Organic samples of red oranges con-

tained similar folate concentrations as conventional pro-

duction, though with higher variability in the folate content

(CV 17 vs 3.5%). Fresh grapefruits and pinapple contained

around 30 and 15 mg folate /100 g, respectively. Pink

grapefruit and yellow grapefruit are good sources of folate,

containing 27–41 mg/100 g, while pineapple contained

lower amounts, 13 mg/100 g.

Retention studies demonstrated that mild technologies,

e.g. mild pasteurisation and high pressure processing (400

or 600 MPa) resulted in good folate retentions, particularly

for red oranges (O90%). Traditional processing as

sterilisation caused 25% loss in red orange juice but similar

folate retention was observed between mild processing and

sterilisation for grapefruit juice and pineapple juice. Folate

content in grapefruit juices did not decrease during

the shelf-life of the product; in pineapple a slight decrease

in folate content was observed.

Values of folate concentrations obtained in the present

study by the quality-controlled microbiological assay

method were higher compared with older data obtained

from various food tables. This finding emphasises the need

to update folates in both fresh fruits and fruit products in all

European countries. In fact, the new data indicate that fresh

oranges and mildly processed orange juice provide similar

folate amounts and one serving of each might contribute

around 100 mg folate (25% of RDA).

Dairy products5-Methyltetrahydrofolate content in five commercial

UHT milks and seven commercial pasteurised milks varied

between 0.8–3.6 mg/100 g and 2.0–4.7 mg/100 g, respect-

ively. Compared with previously published data (Forssen,

Jagerstad, Wigertz, & Witthoft, 2000) the folate values

observed here were in the lower range. This might be due to

low folate content in the raw milk influenced by cattle

feeding or losses of folate owing to the amount of oxygen

dissolved in milk, and level of ascorbic acid in connection to

the applied pasteurisation or UHT technology. In addition,

30 commercial fermented milk products from ten producers

contained between 0.2 and 4.6 mg 5-methyltetrahydrofolate/

100 g. Values are under or close to the levels reported in the

literature, 2–10 mg/100 g (Forssen et al., 2000).

Testing of selected microbial species and strains proved

Bifidobacterium longum, Streptococcus thermophilus and

Propionibacterium freudenreichii subsp. shermanii to

produce folate with Streptococcus thermophilus being the

most dominant producer. The level of 5-methyltetrahydro-

folate differed not only within the species but within the

strains as well. Of three tested Streptococcus thermophilus

strains the most productive strain reached the 5-methylte-

trahydrofolate level seven times higher in comparison with

the weakest producer. Maximum values were reached

within 6–12 h after inoculation, thereafter declining 5-

methyltetrahydrofolate values appeared. A number of

possible stimulants for 5-methyltetrahydrofolate production

were tested for Bifidobacterium longum and Streptococcus

thermophilus, e.g. cystein hydrochloride (1%), sodium

ascorbate (1%), glucose (1%), lactose (2%) and inulin

(1%). However, none of these tested stimulants caused any

significant increase of 5-methyltetrahydrofolate. In cofer-

mentation (37 8C/12 h) with butter starter and the most

productive strains of Streptococcus thermophilus and

Propionibacterium freudenreichii subsp. Shermanii, the

5-methyltetrahydrofolate content 6.3 mg/100 g was reached.

The content found in model samples represents almost

20 times increase in folate content in comparison with

commercial fermented products with low 5-methyltetrahy-

drofolate. In comparison with the analysed commercially

Table 3. Overview of new and refined processing techniques for food with enhanced folate content

Processing technique Applications Folate increases Remarks

Milling Cereal fractionation 10-fold Differences between inner kernel and branfractions

Malting/germination Cereal ingredient forbread, muesli or beer

1 to 3.5-fold

Fermentation Bread making Up to2-fold (yeast) Sour dough fermentation with LAB has no guaran-teed folate enhancing effect, more studies needed

Beer brewing Up to7-fold Folate conc varies between 30 and 180 mg/L in 120EU beers

Milk products Up to 20-fold Depending on starter cultureVegetables Up to 2-fold Depending on starter culture

High pressure or mildpasteurisation

Fruit juices Reduced losses compared with traditional sterilisa-tion, 10 vs 30%

Fortification with folicacid

Wheat flour, milk According to regulations Overages between 10 and 25% are necessary tocompensate for processing losses

M. Jagerstad et al. / Trends in Food Science & Technology 16 (2005) 298–306 305

fermented products having high 5-methyltetrahydrofolate,

the increase is about 40%.

Natural and added folate binding protein (FBP) were

shown to decrease the bioavailability of folates from milk

(Verwei et al., 2003) However, since folate binding proteins

are heat-sensitive they are normally inactivated by heating

above normal pasteurisation. This means that UHT milk and

fermented milk which are processed above normal pasteur-

isation temperatures do not contain any active FBP (Forssen

et al., 2000).

The stability of folic acid fortified milk showed total

folate content of fortified pasteurised milk to contain

466 mg/L of which native folate constituted 46 mg/L. Thus,

of the 478 mg added, 420 mg was retained (Z12% loss).

Total folate content of fortified UHT milk was 402 mg/L, of

which native folate amounted to 40 mg/L, thus of the 478 mg

added folic acid, 362 mg was retained (Z24% loss).

Wigertz, Hansen, Hoier-Madsen, Holm, and Jager-

stad (1996) reported slightly lower losses of 8% for

pasteurisation and 19% for UHT treatment for the native

5-methyltetrahydrofolate form. Possible differences in the

exact pasteurisation and UHT procedures used can explain

such differences.

ConclusionsTable 3 summarises the main results of new and refined

processing techniques for food with enhanced folate

content reported in this project review. Yeast fermentation,

malting, germination and LAB can be combined and

further optimised to potentially enhance the folate content

in bread, vegetables, dairy products and beer by 2–3 fold.

Further research, exploration and development of folate

producing LAB and yeast strains and combinations of

them for food applications should be encouraged. More

needs to be learned about folate forms produced and the

interactions between food matrix and folate producing

microorganisms. The recent trends towards LCMS or

LCMSMS methods for efficient analysis of different folate

forms will enable future fruitful work in this field with

promising applications not only for traditional foods but

also in developing novel foods, and functional foods

including probiotics and GMO.

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