Effect Of Prebiotics on Folate Production By Lactic Acid Bacteria In Milk Fermentation

INTRODUCTION

Folate is a water-soluble B- vitamin which has an essential nutritional function in the human body, especially very important for pregnant women and lactating mothers. The major sources of folate are green leafy vegetables, liver, legumes, egg yolk, wheat germ, milk and milk products, and yeast (Lin & Young, 2000). Folate deficiency is a global issue especially from developing countries, where the low folate intake has been linked with health disorders such as cancer, anemia, cardiovascular diseases, or neural tube defects. More than 60 countries around the world practice mandatory folate fortification programs using synthetic vitamins such as folic acid to ensure the prevention of diseases and disorders related to folate deficiency caused by health and social problems like malnutrition and unbalanced diet (Albuquerque, 2018; Bailey, Rampersaud, & Kauwell, 2003).

Humans and other animals are incapable of synthesizing folate, they get this vitamin from foods or dietary supplements (Laiño, del Valle, de Giori, & LeBlanc, 2014). Apart from having a high cost of production, high levels of synthetic folic acid in the blood will cause a variety of metabolic disorders such as masking symptoms of vitamin B12 deficiency, cognitive impairment, reducing the immune system, and cancer (Fajardo, Alonso-Aperte, Varela-Moreiras, & analysis, 2012).

Bio-fortification with natural folates by selected microorganisms may be an alternative to fortification with synthetic folic acid. Lactic acid bacteria (LAB) and probiotics strains are good folate producers among food-grade microorganisms, and the use of them has replaced the mandatory fortification with folate to prevent deficiencies that are increasing in different populations all over the world (Gangadharan, Nampoothiri, & Technology, 2011; Laiño et al., 2014).

Milk is among the most nutritious food and contains folate-binding protein, hence it is a good fermentation medium to increase folate stability during storage. Fermentation could increase the levels of folate since LAB are widely used in the fermentation process of dairy products; it is known that the folate is very sensitive to heat treatment and folate content in milk is not high, especially after the application of pasteurization or UHT process (Mousavi et al., 2013).

Polysaccharides extracted from plants can apply the potential prebiotic effect, stimulating the growth of beneficial microorganisms, and also promote their metabolic activity regarding the production folate during the fermentation process. Prebiotics are defined as “a selectively fermented ingredient that results in specific changes in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefit(s) upon host health” (Pop et al., 2019). Therefore, combining vitamin-producing microorganisms with prebiotics seems to be an interesting strategy to develop innovative bio-enriched functional foods such as fermented milk.

To the best of our knowledge, there are few reports about the impact of polysaccharides (prebiotics) addition on microbial growth and folate synthesis during milk fermentation. Thus, considering that prebiotics stimulate the growth of LAB, which produce water-soluble vitamins, it is of interest to determine the production of folate could be enhanced when these bacteria are grown in the presence of galactooligosaccharides (GOS) and inulin. This study aims to investigate the effect of two prebiotics (GOS and inulin) on folate production by folate-producing lactic acid bacteria in milk fermentation.

OBJECTIVES

The objectives of this study will be:

1. To select the appropriate strains of lactic acid bacteria which can produce folates
2. Evaluate the effect of GOS and Inulin over folate production and LAB (Lactobacillus delbrueckii ssp. Bulgaricus, Streptococcus thermophilus, and Bifidobacterium) to develop enriched fermented milk
3. Evaluate the ability of LAB to produce folate during milk fermentation containing GOS and Inulin
4. To evaluate the level of folate production by an isolated LAB strain from fermented milk using HPLC method

EXPERIMENTAL DESIGN

1. Flowchart

Experimental analysis of this study will be conducted in Lanzhou University, Nutrition Analysis Laboratory

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• Hypothesis: When LAB (Lactobacillus delbrueckii ssp. Bulgaricus, Streptococcus thermophilus, and Bifidobacterium) are grown in the presence of galactooligosaccharides (GOS) and inulin, production of folate could be increased.
• Independent variables: polysaccharides (GOS and inulin)
• Dependent variables: folate concentration
• Constants: will be starter culture (Lactobacillus delbrueckii ssp. Bulgaricus, Streptococcus thermophilus, and Bifidobacterium), time, and temperature conditions.
• Experimental group: will be consist of milk samples, lactic acid bacteria, and polysaccharides
• Control group: will be consist of milk samples and lactic acid bacteria
• Replication: this experiment will be repeated three times for accuracy
• Milk samples: Pasteurized milk and skim milk powder

2. Microorganisms

• Lactobacillus delbrueckii ssp. Bulgaricus,
• Streptococcus thermophilus and
• Bifidobacterium

3. Chemicals and mediums

• Galactooligosaccharides
• Inulin
• MRS medium
• Semi-skimmed milk
• MRS medium
• Trypticase soy-yeast extract
• L-cysteine
• Standards of folic acid
• Distilled water

4. Consumables and instruments

• Plate agar
• Analytical weights - Kern abs 220-4
• Autoclave
• HPLC
• Automatic pipettes (2-20 μl, 20-200 μl, 200-1000 μl)
• Gas turner
• Orbital shaker incubator Multitron Single
• SPSS Analysis software
• pH meter
• Glass beakers
• Hot water bath
• Refrigerator
• Sterile test tubes (plastic)
• Syringe filters 0.22 μm
• Lab coat

5. Sample collection

The milk samples (Pasteurized milk and skim milk powder) will be collected from a dairy plant and transfer to the Lanzhou University Nutrition Analysis Laboratory for studies. The samples will be stored under manufacturer recommended storage conditions for subsequent experiments.

7. Sample preparation

L. bulgaricus strains will grow in Man Rogosa Sharpe (MRS) broth, S. thermophilus will grow in Man Rogosa Sharpe (MRS) supplemented with trypticase soy-yeast extract (TSYE) broth and Bifidobacterium strains will be grown in MRS broth supplemented with L-cysteine (0.05 g 100 mL-1) and all strains will be grown in reconstituted non-fat powdered milk autoclave at 121 oC for 15 min. and then strains will be anaerobically incubated at 37 oC for 24 h. The inoculum (10-5 cfu mL-1) will be the same for all bacteria strains in every medium.

Prebiotic concentration of 1.0 g 100 mL-1 will be added to the milk and culture media. Culture media and milk, with or without prebiotics, will be collected after 0, 6, 12, and 24 h of bacterial growth, and folate levels will be analyzed by high-performance liquid chromatography (HPLC). All plates will be incubated anaerobically at 37 oC for 72 h.

8. Folate standards

Individual folate standards:

• (6R, S)-5,6,7,8-tetrahydrofolic acid calcium salt (H4-folate),
• (6R, S)-5-methyl-5,6,7,8-tetrahydrofolic acid sodium salt (5eCH3eH4-folate)
• and (6R, S)-5-formyl-5,6,7,8- tetrahydrofolic acid sodium salt (5eHCOeH4-folate)

Standard solutions will be prepared according to the method described by Van den Berg, Finglas, and Bates (Van den Berg, Finglas, Bates, & Nutrition, 1994).

9. Extraction and deconjugation of folates from samples

The extraction, deconjugation, and purification procedures will be carried out under subdued light to prevent photodegradation of folates. Folates from bacterial cultures will be extracted following the procedure described by Pfeiffer, Rogers, and Gregory (Pfeiffer, Rogers, Gregory, & Chemistry, 1997) and Konings (Konings, 1999). The eluted sample will be weighed, and the purified extracts will be kept under refrigeration for no longer than 2 h before they will be placed in the auto-sampler and injected onto the HPLC.

10. High-performance liquid chromatography analysis of folates

The precision of the HPLC analysis including sample extraction, deconjugation, and purification showed recoveries of spiked folates on the samples studied at a level of 50 ng mL1 that ranged from 75 to 100% for H4- folate, from 70 to 99% for 5eCH3eH4-folate and from 80 to 100% for 5eHCOeH4-folate. The coefficient of inter- and intra-assay variation for folate analysis was below 10%. The limits of quantification were 2.34 ng mL1 for H4-folate, 2.67 ng mL1 for 5eCH3eH4-folate and 34.20 ng mL1 for 5eHCOeH4-folate. The folate content was expressed as micrograms per 100 mL of fresh medium in all samples.

11. Statistical analysis

SPSS software package for Windows will be used to test statistical analysis of the data Analysis of variance (ANOVA) of variation in the content of folates amongst the different LAB for a given sampling time, as well as the variation in the content of folates due to the effect of the prebiotics.

12. Research outcome

1. To finish the assigned research topic and publish one academic paper in journals of science.
2. The use of prebiotics with LAB in milk fermentation, it is hoped that will increase the level of natural folate content which is beneficial to human health.

Application: after accomplished this research, it will be recommended and applied to dairy plant

13. Timeline:

• From May to June 2020: I will attend laboratory activities to be familiar with instrument and process
• July: I will experiment with this research and data analysis
• August: I will write a research paper

References

1. Albuquerque, M. A. C. d. (2018). Effect of vegetable by-products on folate production by starter and probiotic microorganisms to develop a bio-enriched fermented soy product. Universidade de São Paulo,
2. Bailey, L. B., Rampersaud, G. C., & Kauwell, G. P. J. T. J. o. n. (2003). Folic acid supplements and fortification affect the risk for neural tube defects, vascular disease, and cancer: evolving science. 133(6), 1961S-1968S.
3. Fajardo, V., Alonso-Aperte, E., Varela-Moreiras, G. J. J. o. f. c., & analysis. (2012). Lack of data on folate in convenience foods: should ready-to-eat products be considered relevant for folate intake? The European challenge. 28(2), 155-163.
4. Gangadharan, D., Nampoothiri, K. M. J. L.-F. S., & Technology. (2011). Folate production using Lactococcus lactis ssp cremoris with implications for fortification of skim milk and fruit juices. 44(9), 1859-1864.
5. Konings, E. J. J. J. o. A. I. (1999). A validated liquid chromatographic method for determining folates in vegetables, milk powder, liver, and flour. 82(1), 119-127.
6. Laiño, J. E., del Valle, M. J., de Giori, G. S., & LeBlanc, J. G. J. J. I. j. o. f. m. (2014). Applicability of a Lactobacillus amylovorus strain as co-culture for natural folate bio-enrichment of fermented milk. 191, 10-16.
7. Lin, M., & Young, C. J. I. D. J. (2000). Folate levels in cultures of lactic acid bacteria. 10(5-6), 409-413.
8. Mousavi, S. S., Moeini, H., Mohamad, R., Dinarvand, M., Ariff, A., Ling, F. H., & Raha, A. R. J. R. i. B. (2013). Effects of medium and culture conditions on folate production by Streptococcus thermophilus BAA-250. 4(6).
9. Pfeiffer, C. M., Rogers, L. M., Gregory, J. F. J. J. o. A., & Chemistry, F. (1997). Determination of folate in cereal-grain food products using tri enzyme extraction and combined affinity and reversed-phase liquid chromatography. 45(2), 407-413.
10. Pop, O. L., Salanță, L.-C., Pop, C. R., Coldea, T., Socaci, S. A., Suharoschi, R., & Vodnar, D. C. (2019). Prebiotics and dairy applications. In Dietary Fiber: Properties, Recovery, and Applications (pp. 247-277): Elsevier.
11. Van den Berg, H., Finglas, P., Bates, C. J. I. J. f. V., & Nutrition, N. r. I. Z. f. V.-u. E. J. I. d. V. e. d. (1994). FLAIR intercomparisons on serum and red cell folate. 64(4), 288-293.