Fermentation of milk whey permeate with different dairy ...

Introduction Microorganisms were obtained from DSM culture collection:

DSM 20273 Prb acidipropionici = A;

DSM 20272 Prb acidipropionici = B;

DSM 4902 Prb freudenreichii subsp. Shermanii = E;

DSM 20535 Prb jensenii = J;

DSM 20276 Prb thoenii = T.

Whey ultrafiltrate (lactose content 46 g/L) was used as a sole fermentation media and substrate. Blank samples without added

microorganisms (O) were also studied as a negative control. Fermentation was performed in 500 mL glass flasks sealed with

cotton plugs and covered with Al foil to protect the broth from drying out. Flasks were placed in the room temperature for 26

days. The samples were not shaken except gentle agitation at the time of sampling.

Sample collection and storage: samples were harvested at different time points aseptically, by carefully mixing the content of

the flasks beforehand. Samples for organic acid content analyses were poured in smaller sealable tubes and kept at -18°C,

for further analysis.

Analytical methods: pH was measured by a pH meter; biomass growth was monitored spectrophotometrically as OD

measurements at 590 nm; content of organic acids was determined by HPLC.

Results

Fermentation of milk whey permeate with different dairy Propionibacteria strains

Unigunde Antone1, Janis Liepins2, Jelena Zagorska1, Ingmars Cinkmanis1

1 Faculty of Food Technology, Latvia University of Life Sciences and Technologies (LLU), Rīgas iela 22, Jelgava, Latvia, [email protected] 2 Institute of Microbiology and Biotechnology, University of Latvia (LU), Jelgavas iela 1, Riga, Latvia

Acknowledgments

The research received funding from the ERDF Post-doctoral Research Support Program (project Nr.1.1.1.2/16/I/001) Research application “Processing of whey into value added products for food industry and agriculture” (Nr.1.1.1.2./VIAA/2/18/307).

Thanks to the Institute of Microbiology and Biotechnology, University of Latvia for cooperation in research.

Also thanks to JSC “Smiltenes Piens” for providing research with raw materials for fermentation.

Utilizing waste products that are generated from the

technological processes is one of the significant problems of

manufacturing companies and environmentalists (Piwowarek et

al., 2018). Manufacture of cheese and curd produces by-

product – whey, which constitutes approximately 90 % of the

raw material. Whey is high in organic matters and amounts,

and thus presents a high potential of environmental pollution

when discarded untreated (Morales et al., 2006). Whey

contains also many valuable ingredients; it has relatively high

lactose content – 4.6 – 5.2 %. However many companies find it

very difficult to process whey and lactose. Often, produced

whey is not processed further, but sold to biogas plants as raw

material at low price. This is usually economically unprofitable,

because transportation of this by-product in large volumes is

relatively expensive. Biotechnology methods can help to find

innovative solutions for more economical use of whey which is

current issue in many Latvian dairy companies. One

perspective way to use the whey is production of organic acid-

based products. Propionic acid is generally regarded as safe

(GRAS) with applications in a wide variety of industries.

Propionate is primarily used for its antimicrobial properties

especially serving as preservatives in agriculture and human

food. Propionibacteria (Prb) can ferment whey sugar lactose

into propionic and acetic acids as their main fermentation

products. However, because of the low concentration of

propionic acid caused partly by strong end-product inhibition,

bio-based propionic acid is more expensive than its chemical

synthesis (Vidra & Nemeth, 2018). However, nowadays

because of the problems associated with increased oil prices

and the benefits of eco-friendly production, biological propionic

acid biosynthesis emerges as a competitor to chemical

synthesis (Ammar & Philippidis 2021; Gonzalez-Garcia et al.,

2017; Alonso et. al., 2015; Vidra & Nemeth, 2018).

In this research we focused on whey ultrafiltration permeate

(ultrafiltrate – UF) fermentation by genetically unmodified (wild)

organisms, choosing five classical Prb strains which are

attributed to dairy Prb subclass. The optimal temperature for

Prb gowth is around 28-30°C, however, for energy saving

purposes the aim of this study was to compare the organic

acid and biomass production efficiency of five classical Prb

strains growing in the room (22±0.5 °C) temperature.

Key words: dairy, Propionic acid bacteria, lactose,

whey permeate, fermentation

References:

Alonso, S., Rendueles, M., Díaz, M. (2015) Microbial production of specialty

organic acids from renewable and waste materials (Review), Critical Reviews in

Biotechnology, Vol. 35(4), pp. 497-513.

Ammar E.M., Philippidis G.P. (2021) Fermentative production of propionic acid:

prospects and limitations of microorganisms and substrates. Appl Microbiol

Biotechnol, 105, pp. 6199–6213; https://doi.org/10.1007/s00253-021-11499-1

Gonzalez-Garcia R.A., McCubbin T., Navone L., Stowers C., Nielsen L.K.,

Marcellin E. (2017) Microbial Propionic Acid Production, Fermentation, 3 (2): 21;

https://doi.org/10.3390/fermentation3020021

Morales J., Choi J.-S., Kim D.-S. (2006) Production rate of propionic acid in

fermentation of cheese whey with enzyme inhibitors, Environmental Progress, 25(3),

pp. 228-234.

Piwowarek K., Lipińska E., Hać-Szymańczuk E., Kieliszek M., Ścibisz I. (2018)

Propionibacterium spp. - source of propionic acid, vitamin B12, and other

metabolites important for the industry. Appl Microbiol Biotechnol, (102), pp. 515–538;

https://doi.org/10.1007/s00253-017-8616-7 Vidra A., Németh Á. (2018) Bio-produced

Propionic Acid: A Review, Periodica Polytechnica Chemical Engineering, 62(1), pp.

57–67; https://doi.org/10.3311/PPch.10805

As a result of acidogenic activity, media total acidity expressed as pH value of the samples with added Prb gradually decreased from the initial pH 6.40, to around pH

4.74 on day 26 (see Fig. 1).

The most rapid decrease in pH occurred during the first 3 days. The slow pH decrease in the remaining fermentation period indicate a decline in the metabolic activity of

microflora due to the end-product inhibition. Decrease of pH value in blank samples most likely can be attributed to the activity of the background microflora (mainly

lactic acid bacteria). The presence of lactic acid bacteria was confirmed by the rapid accumulation of lactic acid content during the first 6 days (see Fig.2c).

The most pronounced increase in biomass during 26-day period was for strains T and J, which also coincides with the highest efficiency of propionic acid production

(see Fig. 2a). However, taking into account the long total fermentation time, the growth of all strains was relatively slow, especially that of the strain B.

Figure 1. Changes of the pH and optical density during UF fermentation by different Prb strains

Also changes of the acetic and lactic acids content in fermentates produced by different Prb strains are given in Figure 2. The maximal propionic acid concentration 3.71

g/L was produced by strain T on day 26, followed by strains J and B (2.50 and 2.49 g/L). The strain T was also the most effective propionic acid producer within 6 and

16-day periods, followed by strain J. The less effective producers or propionic acid within 16-day period were strains A, B and E. Interestingly that strain B propionic acid

production initially was the slowest, but after 6 period its rate increased quite rapidly, surpassing even strains A and E at the end of the fermentation (day 26).

The increase in propionic acid content was gradual, while the acetic acid content increased rapidly in the first 6 days, most likely by metabolical activity of Prb and also

due to the activity of the background microflora (lactic acid bacteria). The further decrease in lactic acid content in samples with added Prb can be explained by the fact

that it was consumed by these microorganisms.

Figure 2. a, b, c - Content of propionic, acetic and lactic acids (g/L±SEM) in the UF permeate broth during Prb fermentation, d – ratio of propionic and acetic acids

0.0

0.7

1.3 1.51

0.0 0.2

1.4

2.49

0.0

0.7

1.4

1.79

0.0

0.8

1.9

2.50

0.0

1.0

2.6

3.71

0.0 0.0 0.0 0.03

d 0 d 6 d 16 d 26

a. Propionic acid, g/L

A B E J T O

0.0

0.7

0.8 0.92

0.0

1.1

1.3 1.25

0.0

1.0

1.2 1.22

0.0

1.1 1.2

1.32

0.0

1.2

1.1 1.13

0.0

1.1 1.0 1.04

d 0 d 6 d 16 d 26

b. Acetic acid, g/L

A B E J T O

2.2

7.7

6.8 6.2

2.2

8.5 7.5

6.5

2.2

7.5 6.9 7.0

2.2

7.5 6.9

6.2

2.2

7.6 7.2 7.3

2.2

9.5 9.4 9.7

d 0 d 6 d 16 d 26

c. Lactic acid, g/L

A B E J T O

0

1.1

1.6 1.6

0 0.2

1.1

2.0

0

0.6

1.1

1.5

0

0.7

1.5

1.9

0

0.8

2.4

3.3

0 0.0 0.0

d 0 d 6 d 16 d 26

d.Propionic/acetic acid ratio

A B E J T O

4.25

4.75

5.25

5.75

6.25

6.75

0 10 20 30

pH

Duration of fermentation, days

A B E J T O

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 10 20 30

OD

, 590 n

m

Duration of fermentation, days

A B E J T O

The main findings of this study are as follows: 1. As expected, all of the strains were able to convert lactose into acid, however, with different efficiency. Production of propionic acid by Prb is strain dependent. The best producers of propionic acid was strains T, J and B in a 26-day period, however as this is a very long time, it should be noted that also the strain E showed relatively high efficiency in a shorter 6-day period. The highest amount of propionic acid was produced by strain T, and it reached 3.7±0.13 g/L within 26-day period. 2. The pore size of the filters (0.01 – 0.1 µm) used in the ultra filtration of the whey does not provide 100% sterility of the raw material. The presence of background microflora in whey permeate most likely are lactic acid bacteria that can be either as natural microflora survived (NSLAB) during milk pasteurization or inoculated starter cultures. 3. The raw material used in fermentation does not have to be pasteurized, provided that suitable strains of Prb bacteria are used and that the raw material is not contaminated with other undesirable microflora (secondary contamination) or bacteriophague infection. Although the presence of lactic acid in the product is desirable, the lactic acid bacteria or their metabolites in the raw material may affect the growth of Prb, which would be worth exploring in future studies.

Methodology

Vidra & Nemeth, 2018