ARCHIVOS LATINOAMERICANOS DE NUTRICIÓN Vol.

63 Nº 1, 2013
Órgano Oficial de la Sociedad Latinoamericana de Nutrición

Viability of probiotic Lactobacillus casei in yoghurt: defining

the best processing step to its addition
Nataly Simões Bandiera, Isadora Carneiro, Alisson Santana da Silva, Edson Renato Honjoya, Elsa Helena
Walter de Santana, Lina Casale Aragon-Alegro, Cínthia Hoch Batista de Souza

Universidade Norte do Paraná, UNOPAR, Londrina, PR, Brasil.

SUMMARY. Probiotics are live microorganisms ca- RESUMO. Viabilidade de Lactobacillus casei probiótico em
pable of producing beneficial effects on its host when iogurte natural: definição da melhor etapa de processamento
consumed in adequate amounts. To exert these effects, para sua adição. Probióticos são microrganismos capazes de pro-
foods must contain probiotic microorganisms in popu- duzir efeitos benéficos sobre seu hospedeiro, quando consumidos
lations above 106 CFU / g or mL throughout its shelf vivos e em quantidades adequadas. Para que exerçam esses efei-
life. One of the strategies to ensure high population of tos, os alimentos probióticos devem conter tais microrganismos
probiotics in fermented milk is to add them during or em populações acima de 106 UFC/g ou mL, durante toda sua va-
after the fermentation process separately from the star- lidade. Uma das estratégias para garantir a alta população dos pro-
ter cultures. The objective of this study was to investi- bióticos em leites fermentados é adicioná-los durante ou após o
gate the behavior of the probiotic microorganism processo de fermentação separadamente das culturas starter.
Lactobacillus casei added to yoghurt in different stages Assim, o objetivo deste trabalho foi verificar o comportamento
of production. Yoghurts with L. casei were produced do microrganismo probiótico Lactobacillus casei adicionado em
at different stages: before addition of starter (Strepto- iogurte natural, em diferentes etapas do processo de produção.
coccus salivarius subsp. thermophilus and Lactobaci- Foram produzidos iogurtes com adição de L. casei em diferentes
llus delbrueckii subsp. bulgaricus), added together with etapas: antes da suplementação com o starter (Streptococcus sa-
this culture and at the end of fermentation. Yoghurt wi- livarius subsp. thermophilus e Lactobacillus delbrueckii subsp.
thout probiotic added was produced as a control. The bulgaricus), juntamente com essa cultura e após o término da fer-
products were stored at 4 °C and analyzed after 1, 7, mentação. Além disso, um iogurte controle (sem probiótico) foi
14 and 21 days of storage. In these periods, the popu- produzido. Os produtos foram estocados a 4°C e analisados após
lations of probiotic and starter cultures were enumera- 1, 7, 14 e 21 dias de armazenamento. Nesses períodos, foram enu-
ted and the parameters pH and acidity were analyzed. meradas as populações das culturas probiótica e starter e avaliados
The results were evaluated using analysis of variance os parâmetros físico-químicos (pH e acidez). Os resultados obti-
and Tukey's test, both at 5% significance level. L. casei dos foram avaliados através de análise de variância e teste de
remained viable in populations of more than 108 CFU Tukey, ambos ao nível de 5% de significância.L. casei manteve-
/ g during 21 days of storage, which is suitable to define se viável e em populações superiores a 108 UFC/g durante os 21
the formulations as probiotics. When the different sta- dias de armazenamento, suficientes para que as formulações fos-
ges of the addition of probiotics in yoghurts were eva- sem definidas como probióticas. Quando as diferentes etapas de
luated there was no statistical difference between the adição do probiótico aos iogurtes foram avaliadas, com exceção
formulations (p<0.05) for populations of L. casei ex- do 1º dia, não houve diferença estatística entre as formulações
cept for the first day of storage. (p<0,05) para as populações de L. casei.
Key words: Yoghurt, Lactobacillus casei, fermenta- Palavras chave: Iogurte, Lactobacillus casei, fermentação, via-
tion, viability. bilidade.

INTRODUCTION above 106 CFU / g during the shelf life of the product (2).
According to Fuller (3), there are three possible mecha-
Probiotics are live microorganisms which nisms of action of probiotics. The first refers to the elimi-
when consumed in adequate amounts confer be- nation of pathogenic microorganisms through the
neficial health effects on the host (1). However, formation of compounds with antimicrobial activity. The
to exercise these function probiotic foods must second mechanism refers to changes in the microbial me-
contain viable probiotic cultures in populations tabolism by increasing or decreasing the enzymatic func-

58
 VIABILITY OF PROBIOTIC LACTOBACILLUS CASEI IN YOGHURT 59

tions. The third mechanism is based on stimulation of production by L. delbrueckii subsp. bulgaricus during
host immunity by increasing the levels of antibodies. storage of yoghurt, which is so-called post-acidifica-
In addition to controlling the intestinal tract, there tion, is one of the factors known to affect the viability
are other benefits of probiotic cultures to the health of of probiotic microorganisms in these products (7).
the host, such as stabilizing the intestinal microbiota Thus, due to the production of inhibitory substan-
after the use of antibiotics; promoting gastrointestinal ces by yoghurt cultures (starter) during the fermenta-
resistance against colonization of pathogens; decrea- tion, it is important to search for each probiotics the
sing the population of pathogens through the produc- best moment to its addition during processing of yog-
tion of acetic and lactic acids, bacteriocins and other hurt. It is known that the addition of probiotic micro-
antimicrobial compounds; increasing the lactose diges- organism concomitantly with the starter culture or
tion in lactose-intolerant individuals; stimulating the after fermentation may interfere its viability (10),
immune system; relieving constipation and increasing which demonstrates the importance of correct timing
absorption of minerals and vitamins (4). Other benefi- of addition of each strain.
cial effects are also attributed to probiotic cultures: re- The objective of this study was to investigate the
duced risk of colon cancer, cardiovascular disease behavior of the probiotic microorganism Lactobacillus
prevention, reduction of plasma cholesterol, antihyper- casei added at different stages of the production of
tensive effect, reducing the activity of ulcerative Heli- yoghurt, enabling the selection of the best stage of its
cobacter pylori, control of ulcerative colitis caused by addition in order to obtain a product with adequate po-
rotavirus and Clostridium difficile, preventing uroge- pulation of this culture.
nital infections, as well as inhibitory effects on the mu-
tagenicity (5). A number of genera of bacteria are used MATERIALS AND METHODS
as probiotics, including L. casei, which has proven be-
nefits to human (2). For the production of fermented Materials
milk the conventional yoghurt bacteria L. delbrueckii For the production of yoghurt, UHT milk (Lider,
subsp. bulgaricus and S. salivarius subsp. thermophilus Lobato, Brazil), skimmed milk powder (Molico, Nes-
also known as starter culture are added to promote the tle, Araçatuba, Brazil), starter culture Streptococcus
fermentation process (6, 7). The starter bacteria have a salivarius subsp. thermophilus and Lactobacillus del-
symbiotic relationship during its multiplication in the brueckii subsp. bulgaricus (YO-MIXTM 496 LYO 100
milk. The beginning of fermentation (acidity < 20 °D) DCU, Danisco, Dangé, France) and probiotic Lacto-
favors the development of S. salivarius subsp. thermo- bacillus casei (431®, Christian Hansen, Valinhos, Bra-
philus, which is stimulated by certain amino acids (es- zil) were used.
pecially valine) produced by L. delbrueckii subsp. Preparation of probiotic and starter cultures
bulgaricus, causing an increase in acidity. In this phase, The inoculums of probiotic and starter cultures
occurs the release of formic acid by S. salivarius subsp. were prepared separately, one day before the produc-
thermophilus, which stimulates the growth of L. del- tion of yoghurt. To do so, the skimmed milk was re-
brueckii subsp. bulgaricus. Temperatures near 46 °C is constituted in water at 80 °C and cooled to 42°C.
unfavorable for the growth of S. salivarius subsp. ther- Subsequently, 2% of the lyophilized cultures were
mophilus, favoring the rapid development of L. del- added to the reconstituted skimmed milk. The mixture
brueckii subsp. bulgaricus trains with production of was incubated at 42 °C until pH 4.8 was reached. All
acetaldehyde, which is the main responsible for the the inoculums were stored at 4 °C for 12-14 h until the
characteristic aroma of yoghurt (8, 9). production of yoghurt.
Due to the metabolism of the strains of S. salivarius Production of yoghurt
subsp. thermophilus and L. delbrueckii subsp. bulga- Four yoghurt formulations were prepared: L1) wi-
ricus during yoghurt manufacture, the viability of pro- thout probiotic supplementation; L2) supplementation
biotic strains added to these products may be impaired. with the probiotic microorganism 1h before the addi-
The combination of probiotic strains with yoghurt cul- tion of the starter culture; L3) supplementation with
tures results in decreased viability of the probiotic du- the probiotic microorganism added together with the
ring the shelf life of the product (10). The lactic acid starter culture; and L4) supplementation with the pro-
60 BANDIERA et al.

biotic microorganism after the fermentation of milk by results were expressed as colony forming units per
starter culture, as soon as pH 4.8 was reached. gram of product (log CFU / g).
The UHT milk was heated to 40 °C and added of Physicochemical analysis
4% skimmed milk powder to increase the dry matter The pH of the samples was determined by direct
and consequently improve the consistency of the yog- measurement using digital potentiometer TEC-2 (Tec-
hurt. The mixture was heat treated at 90 °C for 3.5 min nal, Piracicaba, Brazil). The titratable acidity was de-
to both destroy microorganisms which could compete termined by titrating the sample with 0.1 N NaOH
with the cultures tested and promote the denaturation solution (Merck, Darmstadt, Germany) in the presence
of whey proteins, which reduces the contraction of the of phenolphthalein indicator (12).
coagulum and consequently reduces syneresis. After Statistical analysis
cooling to 42 °C, the cultures (probiotic and / or star- The results were analyzed by analysis of variance
ter) were added to the different formulations, accor- (ANOVA) and Tukey's test, both at 5% significance
ding to described above. The mixture was incubated level, using the Statistica software (13).
at 42 °C until reaching pH 4.8. For this purpose, the
pH was checked during the fermentation process every RESULTS
30 min for two hours and every 15 min until pH 4.8.
After this step, the product was manually homogeni- During the fermentation time, pH values of all pro-
zed, cooled and kept at 4 °C. All formulations were ducts decreased significantly (p<0.05). Initially, the
produced in triplicate. yoghurts L1 and L4 showed higher pH values (p<0.05)
Storage and sampling periods than the other formulations. The formulation L2, in
The yoghurts were stored for 21 days at 4 °C until which the probiotic microorganism was added before
the time of analysis. Microbiological (probiotics and the starter culture, presented the lowest initial pH
starter counts) and physicochemical parameters (pH (p<0.05) and L3 showed an intermediate pH value
and acidity) of the samples were performed in tripli- (p<0.05). From 90 min of fermentation, there was no
cate after 1, 7, 14 and 21 days of storage. statistical difference (p>0.05) between the formulations.
Microbiological analysis Figure 1 shows the pH values of yoghurt during the
To carry out the microbiological analyzes, 10 g fermentation time (255 minutes). From the 1st and the
sample was homogenized into sterile bags with 90 mL 21th day of refrigerated storage, the pH decreased for
of sterilized saline solution (0.95% w/v) to obtain the all the formulations (p<0.05). This reduction was gre-
initial dilution (10-1). From this dilution, a number of ater in L2 and L3, in which L. casei was added before
decimal dilutions were prepared using the same diluent. and concomitantly with the addition of starter culture,
For enumeration of Lactobacillus casei, the dilutions respectively. The pH of yoghurt L1 was higher than
were plated in depth in MRS agar (Oxoid, Basingstoke, L2 and L3 (p<0.05), since this sample was not added
UK) supplemented with 1.5 g / L of bile salts. The pla- of L. casei. In contrast, L4 presented pH values similar
tes were incubated for 72 h at 37 °C under aerobic con- to those observed in L1 (p>0.05), even containing L.
ditions. For enumeration of Streptococcus salivarius casei in the formulation.
subsp. thermophilus, dilutions were plated in depth in Figure 2 shows the pH and titratable acidity of yog-
MRS agar (Oxoid) containing 10% lactose (Oxoid) and hurt L1, L2, L3 and L4 stored at 4 °C for 21 days.
the plates were incubated for 48 h at 37 °C under aero- There was no statistically significant difference bet-
bic conditions. For the enumeration of Lactobacillus ween all the formulations after the 1st and 7st days of
delbrueckii subsp. bulgaricus, the decimal dilutions storage (p>0.05). However, at 14 and 21 days of sto-
were seeded in depth in MRS agar (Oxoid) acidified to rage, statistically significant difference was observed
pH 5.4 with glacial acetic acid. In samples containing when formulations L1 and L4 where compared to L2
L. casei, the population of L. delbrueckii subsp. bulga- and L3 (p<0.05).
ricus was obtained by subtracting the population of L. Figure 3 presents the microbiological results obtai-
casei from the total number of Lactobacillus counted ned for starter culture. For populations of L. delbrueckii
in acidified MRS agar. All microbiological analyzes subsp. bulgaricus, the formulation L1 showed lower
were performed according to Lima et al. (11) and the counts than the other formulations (p<0.05), ranging
 VIABILITY OF PROBIOTIC LACTOBACILLUS CASEI IN YOGHURT 61

FIGURE 1. pH values of yoghurt L1 (without addition of L. casei),

L2 (L. casei before addition of the starter culture), L3 (L. casei
added together with the starter culture) and L4 (addition of
L. casei after fermentation) during the fermentation time.

FIGURE 3. (a) Population of Lactobacillus

delbrueckii subsp. bulgaricus and (b) Streptococcus
salivarius subsp. thermophilus in yoghurt L1
(without addition of L. casei), L2 (L. casei before
addition of the starter culture), L3 (L. casei added
together with the starter culture) and L4 (addition
of L. casei after fermentation) during 21 days of
FIGURE 2. pH and titratable acidity values of yoghurt L1 (without refrigerated storage (4°C).
addition of L. casei), L2 (L. casei before addition of the starter
culture), L3 (L. casei added together with the starter culture)
and L4 (addition of L. casei after fermentation) during 21 days
of refrigerated storage (4 °C).

between 6.2 ± 0.5 and 5.6 ± 0.3 log CFU / g

at the 1st and 21th day of storage, respecti-
vely. In contrast, the populations of L. del-
brueckii subsp. bulgaricus found in
formulations containing L. casei were signi-
ficantly higher (p<0.05), reaching counts of
9.3 ± 0.2, 8.8 ± 0.1 and 8.1 ± 0.4 log CFU /
g at 21th day for L2, L3 and L4, respectively
(Figure 3a). Despite minor variations, there
FIGURE 4. Population of Lactobacillus casei in yoghurt L2
was no statistically significant difference in (L. casei before addition of the starter culture), L3 (L. casei added
the populations of S. thermophillus for all together with the starter culture) and L4 (addition of L. casei after
the yoghurts studied (p>0.05) (Figure 3b). fermentation) during 21 days of refrigerated storage (4 °C).
62 BANDIERA et al.

The probiotic populations observed in L2, L3 and storage at 4 °C, ranging from 4.93 (day 1) to 4.32 (day
L4 are presented in Figure 4. A higher population of 19), similar to that observed in the present study for the
L. casei was observed in formulation L2, when com- formulations with L. casei, particularly in L2 and L3.
pared to the formulation L3 (p<0.05) at 1st and 7st days. In contrast, the results observed in formulation L4, re-
When L2 and L3 where compared on days 14 and 21, vealed a manufacture process that may minimize the
no statistical difference was observed (p>0.05). The excessive post-acidification during the shelf life of the
formulation L4 presented the lowest populations of L. product. Although the metabolic activity of lactic acid
casei, differing from the others during 7 days of refri- bacteria be reduced under refrigeration, the final product
gerated storage (p<0.05). At the end of refrigerated may suffer a phenomenon called post-acidification, that
storage (day 21), no statistically significant difference is the decrease of pH during storage due to persistent
was observed among L2, L3 and L4 (p>0.05). Regar- metabolic activity of the starter culture added to the
ding the time of addition of the probiotic microorga- product (mainly L. delbrueckii subsp. bulgaricus) (18).
nism it was found that, except for the first day, there The presence of L. casei did not affect L. delbruec-
was no statistical difference in the populations of L. kii subsp. bulgaricus populations, since populations
casei for all formulations during the whole storage pe- above 8 log CFU / g were detected in yoghurt at 21th
riod (p>0.05). day of storage. Between the 1st and the 7th day of sto-
rage, there was a decrease in the populations of L. casei
DISCUSSION for all formulations probably due to stress caused by
the process and / or low temperature. Between the 7th
The addition of L. casei at the end of the fermenta- and 14th day there was an increase in the populations
tion process in L4 resulted in pH values similar to L1 of L. casei probably due to adaptation of the microor-
(p>0.05) since the yoghurt was cooled promptly after ganism to the matrix that constitutes the product. At the
addition of the culture. Thus, the low temperature con- last week's storage of yoghurt, there was a reduction of
tributed for decreasing the metabolism of L. casei in these populations probably caused by adverse condi-
L4, which resulted in pH values higher than those ob- tions, such as lack of nutrients and low pH. Although
served for other probiotic formulations. some variations and reductions were observed during
The reduction in pH and increase in titratable aci- the storage period, L. casei populations remained al-
dity values observed during the whole storage period ways above the minimum recommended for a probiotic
is a natural process caused by the continuous produc- food (106 CFU / g). Therefore it was possible to consi-
tion of lactic acid and other organic acids by starter der the formulations L2, L3 and L4 as probiotic yog-
culture added to yoghurt. Also, the presence of pro- hurts established by the Brazilian regulation (19).
biotic microorganism contributed to low pH values in Similarly, Kristo et al. (20) produced probiotic
L2 and L3, since L. casei are capable to produce lactic fermented milk containing Lactobacillus paracasei
acid from hexoses (14, 15). B117 in co-cultured with Streptococcus thermophilus
Thamer and Penna (16) produced 12 functional and Lactobacillus bulgaricus Y4.10 and Y6.15. The
dairy beverages formulations containing Lactobacillus authors found that populations of L. paracasei remai-
acidophilus and Bifidobacterium and a prebiotic ingre- ned with counts of 106 CFU/mL during 21 days of
dient. After completion of fermentation and cooling, the storage at 4 °C.
pH ranged between 4.72 and 4.83. The authors high- Regarding the time of addition of the probiotic mi-
lighted that by starting the cooling at pH 4.8, it preven- croorganism to the yoghurt, different results from
ted excessive lowering of pH, which when less than 4.0 those observed in this study were verified by Lanka-
may decrease the protein hydration and the clotting pro- puthra and Shah (6), which produced yoghurt by fer-
perty, resulting in separation of the whey. Kempka et al. menting milk initially only with probiotic
(17) developed a fermented milk drink peach flavor microorganisms L. acidophilus and B. longum for two
with the addition of a culture consisting of Streptococ- hours. After this period, when such microorganisms
cus thermophilus, Lactobacillus bulgaricus, Lactoba- were in the final adjustment phase or in early log
cillus acidophilus and Bifidobacterium. The authors phase, the cultures of yoghurt were added. After six
found that the pH values decreased significantly during weeks of storage, the authors observed that the pro-
 VIABILITY OF PROBIOTIC LACTOBACILLUS CASEI IN YOGHURT 63

biotics counts were approximately one log cycle gre- technological properties. J Biotechnol. 2000; 84:
ater than when these microorganisms were added be- 197-215.
fore the yoghurt cultures as compared to the yoghurt 11. Lima KGC, Kruger MF, Behrens J, Destro MT, Land-
with cultures added concurrently. graf M, Franco, BDGM. Evaluation of culture media
for enumeration of Lactobacillus acidophilus, Lacto-
bacillus casei and Bifidobacterium animalis in the pre-
CONCLUSION sence of Lactobacillus delbrueckii subsp. bulgaricus
and Streptococcus thermophilus. Food Sci Technol.
The probiotic microorganism Lactobacillus casei 2009; 42: 491-95.
remained viable, in populations above 8 log CFU / g 12. Association of Official Agricultural Chemists. Official
during the refrigerated storage of yoghurt. Regarding Methods of Analysis. 15 ed. Washington, 1995. 109.
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