Whey is a yellow-green liquid by-product in cheese manufacturing, remaining after the
precipitation and removal of fat and casein from whole milk and represents about 85-90% of the milk
volume. The main composition of dried whey is 70% lactose, 9-14% protein, and 9% ash (3,5,7,10).
The worldwide dairy industry generates over 80 million tons of whey each year. Whey represents an
important environmental problem due to the high volumes produced and its high organic matter
content, exhibiting a BOD of 30000-50000 ppm and COD of 60000-80000 ppm, with lactose being
largely responsible for the high BOD and COD (60 g oxygen per liter) (2,4,7,11,13). Thus, whey is
particularly suitable for the production of SCP using lactose-utilizing yeast (13).
SCP production technologies arose as a promising way to solve the problem of worldwide protein
shortage. They evolved as bioconversion processes which turned low-value by-products, often wastes,
into products with added nutritional and market value and since SCP belongs to one of the cheapest
protein products in the market, its production is profitable (6,12).
As compared with plants and animals for providing proteins for food or feed, large-scale industrial
production of microbial biomass for the same use has great characteristic advantages such as:
Microorganisms in general have a high rate of multiplication and a high protein content (30-80%
protein in terms of dry weight); They can utilize a large number of different low cost carbon sources
including waste materials; Production installations occupy limited areas and give high yields and
except for algae microbial production is independent of seasonal and climatic variations and therefore
more easy to plan (1,8,13).
A special problem with SCP products for human consumption is the nucleic acid content. High
content of nucleic acids causes no problems to animals since uric acid is converted to allatonin which
is readily excreted in urine. Some practical methods for solving this problem for human use of SCP arealkaline or acidic hydrolysis and activation of endogenous RNA-ases (usually by brief heat treatment
at 64°C for 30 min) (1,8).
Microbial biomass is broadly used as fermentation starter cultures for food and beverage
fermentations, waste treatment processes and agricultural inoculants; as a source of protein for human
food, because it is often odorless and tasteless; as animal fodder and as functional foods, because it has
flavor, fat and water binding property, dispersing action, whipping and foaming action and extrusion
properties (8,13).
Several processes such as Kiel process in Germany and Vienna process in Austria have been
developed for the utilization of lactose in whey to produce SCP. Some of more successful processes
were operated by Bel industries in France. The Bel process was developed with the aim of reducing
the pollution load of dairy industry waste, while simultaneously producing a marketable protein
product (13). While most organisms do not grow on lactose as a carbon source, strains of
Kluyveromyces marxianus readily grow on lactose (4). A number of plants are operated using
Kluyveromyces lactis or K. marxianus (formerly K. fragilis) to produce protein, Protibel, which is used
as a nutritional supplement for both human and animal consumption (13).
The aim of the present work was to investigate the potential and effectiveness of using UF whey
as a substrate under batch fermentation for the production of single cell protein by the yeast
kluyveromyces marxianus. The information would be useful for the development of a cost effective
process in a large industrial scale to produce protein.
2. Materials and Methods
2.1. Whey preparation
Cheese whey was obtained from the Ramak Dairy Factory in Shiraz. It was drawn from the pipe
into 2 L plastic containers. The containers were sealed and transported to the Dept. of Food Science
Laboratory at Shiraz University in Shiraz, where they were stored in a freezer at -20°C until required.
Some characteristics of the cheese whey used in this study are presented in Table 1. Prior to
fermenting process of cheese whey it was allowed to completely thaw at room temperature for 24 h
(4,5).
To reach the highest biomass yield, 0.8 g/l ammonium sulphate as nitrogen source was added to
whey (9,12). Two liters of raw cheese whey was pasteurized in a bottle (4,5,13). The pasteurization
technique included heating the whey to 65°C for 30 min, cooling it to 0°C for 30 min and letting it to
stand at room temperature (25°C) for 24 h for any spore to germinate. The process of heating, cooling
and standing at room temperature was repeated three times to destroy any vegetative or spore cells
present in the whey (4,5).
2.2. Inoculum preparation
Lyophilized yeast strain K. marxianus (PTCC 5193) was obtained from the Persian Type Culture
Collection, Tehran, Iran. A small amount of a pellet of K. marxianus was dissolved in a 7 mL
sterilized growth medium tube containing 1% yeast extract, 2% peptone and 2% dextrose. The tube
was placed at 30°C for 48 h to activate the yeasts (4,5).
A loop of this solution was streaked on an agar medium, containing 1% yeast extract, 2% peptone,
2% dextrose and 2% agar in a Petri dish (3 Petri dishes were used). The Petri dishes were then placed
in an incubator at 30°C and left until visual growth appeared (after 72 h). Then they stored in
refrigerator at 4°C until needed. The yeast colonies were then scooped from the surface of the agar
into 80 mL pasteurized cheese whey in the sterilized Erlenmeyer flask. The Erlenmeyer flask was then
capped with cotton and mounted on a shaker (4). The shaker was operated at a speed of 170 rpm for
48 h at 35°C. The number of yeast cells in inoculum was measured to insure the effectiveness of the
conditions performed on the shaker.SCP production
A 1 L flask was filled with 675 mL pasteurized whey. Then, 75 mL of the inoculum (10% by
volume) were added as recommended by Ghaly et al. (4). The flask content was mixed thoroughly and
then distributed in 15 flask (each 50 mL). Then flasks were shaked for 96 h. The shaker was operated
at a speed of 200 rpm and 35°C temperature. The optimum temperature for K. marxianus propagation
is in the range of 30–35°C (4). Flasks were taken out at 0, 6, 18, 24 and 96 h in 3 replicates.
Immediately after the termination of the fermentation process, the flasks were put in water bath at
100°C for 10 min to deactivate the yeasts and interrupt the process. Finally samples were transferred
to centrifugation unit to recover the yeast biomass from the spent medium.
2.4. Analysis of cheese whey
The chemical analysis of whey was performed using AOAC test methods. Protein content was
determined by microKjeldahl method with 6.25 conversion factor.
2.5. Yeast cells number measurement of the inoculum
1 mL of the inoculum was diluted in 5 tubes each contained 9 mL saline solution consisted of
0.9% NaCl salt. The dilution rate continued to 10-5. Then 0.5 mL solution of both 10-4 and 10-5 dilution
tubes were differential cultured on agar yeast-peptone-dextrose (YPD) growth medium in 2 replicates.
The plates were placed in incubator at 35°C for 48 h until the colonies appeared and were countable.
2.6. Biomass yield measurement
After the termination of the process, 40 mL of each sample were transferred to centrifugation
tubes in triplicate and centrifugated at 4000 rpm (9). The biomass then was dried in vacuum oven at
60°C for 8 h and then weighed. The results are shown in Figure 1.
2.7. Analysis of biomass protein
The protein analysis of biomass was performed using AOAC test methods. Protein content was
determined by microKjeldahl method with 6.25 conversion factor. The results are shown in Figure 1.
3. Results and Discussion
3.1. Whey characterization
The physical and chemical characteristics of ultra filtration permeate cheese whey are given in
Table 1. The initial pH value of the cheese whey used in this study was 7.2, however the optimum pH
for the growth and survival of K. marxianus is between 4.0 and 5.0 (4). It has also been recognized
that keeping the pH at about 4.5 eliminates possible contamination by lethal bacteria that grow at pH
above 6.0 (4). Thus, in this study, pH of the medium was adjusted to 4.5 by the addition of 1 N HCl
solution (4).
Since the protein content of whey was low, no difficulties related to precipitation of protein during
preparation were encountered and therefore UF cheese whey is a more suitable substrate for SCP
production than other wheys.
3.2. Inoculum cell number
The colonies developed from the samples showed the elevated concave, smooth appearance and
creamy color of K. marxianus. Direct plate count at 10-4 dilution was impossible since the number
colonies exceeded 300. But at the 10-5 dilution plates were countable and yeast cells number (theaverage of 2 replicates) was 142, thereby allowing to calculate cell number per mL of inoculum was as
follows: 142×105×2= 24800000 cell/mL
This result indicates that the growth conditions were suitable and as no other kind of colony was
observed, no contamination had been occurred and the inoculum was pure.
3.3. Biomass yield and single cell protein production
Data obtained from the amount of biomass yield corresponds with that of protein produced during
fermentation in this study. About 82% of total protein was produced in the first 18 h of 96 h
fermentation, which can be an indication of the exponential phase of the yeast growth. Total amount of
protein in 96 h was measured 38.34% which is considerable and represents UF cheese whey suitable
for protein production under conditions treated.
4. Conclusions
With regards to the results obtained from this work, ultra filtration permeate cheese whey is a
proper substrate for single cell protein production under conditions provided in this study, however,
for profitable production, interruption of the process in the first 18 h, in which 82% of total protein
would be produced, can be useful. Also, further studies should be done to investigate the nucleic acid
content and find some ways to reduce it to permitted levels. Finally, cost effective SCP process can be
performed in an industrial scale and the product can be consumed instead of expensive proteins
present in the market (12).Acknowledgements
Ultra filtration permeate cheese whey was kindly provided by Ramak Dairy Factory, 20th km in
Booshehr road, Shiraz, Iran. This work has been financed by the Kar Afarini Center of the School of
Agriculture, Shiraz University. We are also grateful to Professor Aminlari and staffs of Dept. of Food
Science & Technology, Shiraz University for their useful help.
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