Probiotic stability of yoghurts containing Jerusalem artichoke inulins during refrigerated storage

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Probiotic stability of yoghurts containing Jerusalem artichoke inulins during refrigerated storage Tatdao Paseephol, Frank Sherkat* School of Applied Sciences, Food Science, RMIT University, GPO Box 2476V, Melbourne, Vic. 3001, Australia

A R T I C L E I N F O

A B S T R A C T

Keywords:

Experimentally prepared Jerusalem artichoke inulins (JAI) were compared with two com-

Jerusalem artichoke

mercial chicory root inulins for their prebiotic potentials in media broth model and

Inulin

growth-sustaining ability in non-fat yoghurts. Experimental yoghurts were made with

Yoghurt

12% reconstituted skim milk (RSM) supplemented with 4% inulin powders, inoculated with

Probiotic

mixed cultures of Lactobacillus casei LC-01, Streptococcus thermophilus and Lactobacillus del-

Prebiotic

brueckii subsp. bulgaricus (1:0.5:0.5 based on supplier’s recommendation) and incubated overnight at 37 C. Non-supplemented yoghurt was prepared from 16% RSM and used as control. The survival and acidifying activity of lactic and probiotic cultures in all yoghurts were investigated on weekly intervals during the shelf life of 28 days at 4 C. Incorporation of JAI resulted in improved viability of LC-01, maintaining >7.0 log CFU/g during cold storage but did not affect the viability of yoghurt bacteria in comparison with the control.  2009 Published by Elsevier Ltd.

1.

Introduction

Consumers across the world are becoming more interested in foods with health promoting features as they gain more awareness of the links between food and health. Among the functional foods, products containing probiotics are showing promising trends worldwide. Probiotics such as Lactobacillus and Bifidobacterium spp. are bacterial members of the normal human intestinal flora (Tamime et al., 2005) that exert several beneficial effects on human health and well-being through production of short-chain fatty acids and improve the intestinal microbial balance, resulting in the inhibition of bacterial pathogens, reduction of colon cancer risk, improving the im-

mune system and lowering serum cholesterol levels (Saarela et al., 2002). Probiotics are recognised for their applications in dairy products, particularly yoghurts and the market for these products is still rising. In Australia, yoghurt consumption has increased steadily from 5.3 kg per capita in 2000 to 6.8 kg in 2007 (Dairy Australia, 2007), and the probiotic yoghurt constitutes the largest share in the probiotic dairy foods market, representing 82% of the total market volume (Anon, 2003). To achieve the claimed health benefits, one of the most important requirements for manufacturing and marketing of probiotic yoghurt is to maintain a high number of probiotic organisms P6 log CFU/g at the point of

Abbreviations: BB, Bifidobacterium bifidum; CFU, colony forming unit; DMRT, Duncan’s Multiple Range test; DP, degree of polymerisation; INY, chicory inulin-containing yoghurt; JA, Jerusalem artichoke; JAI, Jerusalem artichoke inulin; JAY, JAI-containing yoghurt; LA, Lactobacillus acidophilus; LB, Lactobacillus delbrueckii subsp. bulgaricus; LC, Lactobacillus casei; LHSMP, low-heat skim milk powder; MRS, de Man–Rogosa–Sharpe; NFCY, non-fat control yoghurt; OD, optical density; OF, oligofructose; OFY, oligofructose-containing yoghurt; SD, standard deviation; ST, Streptococcus thermophilus; TA, titratable acidity. * Corresponding author: Tel.: +61 3 9925 2130; fax: +61 3 9925 5241. E-mail address: [email protected] (F. Sherkat). 1756-4646/$ - see front matter  2009 Published by Elsevier Ltd. doi:10.1016/j.jff.2009.07.001

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consumption (Lourens-Hattingh & Viljoen, 2001). However, in commercial products various probiotic lactobacilli and bifidobacteria show a decline in their viability during product’s shelf life (Hull et al., 1984; Medina & Jordano, 1994). Several factors are responsible for the viability of these organisms e.g. the strains used, culture conditions, antagonism among cultures present, storage time and temperature, initial counts, hydrogen peroxide and oxygen contents in the medium, and the amount of organic acids in the product (Medina & Jordano, 1994; Shah, 2000). Probiotic organisms especially bifidobacteria grow slowly in milk due, in part, to their lack of proteolytic activity, thus requiring the incorporation of essential growth factors such as peptides and amino acids to enhance their growth (Klaver et al., 1993). Considerable studies have been conducted to stimulate the growth of probiotic bacteria during yoghurt fermentation and to improve their survival until the use-by-date, by supplementing yoghurt milk with growth factors such as vitaminenriched protein hydrolysate, amino nitrogen and whey protein concentrate (Akalin et al., 2007; Amatayakul et al., 2006; Dave & Shah, 1998; Kailasapathy & Supriadi, 1996; Klaver et al., 1993). Co-culturing with proteolytic yoghurt bacteria i.e. Lactobacillus delbrueckii subsp. bulgaricus (LB) and Streptococcus thermophilus (ST) also enhances the growth and viability of probiotics and helps to reduce fermentation time (Dave & Shah, 1998). A recent approach is to incorporate prebiotic substrates to support the growth and activity of probiotics (Akalin et al., 2004; Bruno et al., 2002; Desai et al., 2004; Shin et al., 2000). Products containing a combination of prebiotics and probiotics are known as synbiotics (Gibson & Roberfroid, 1995). The most studied prebiotic substances are fructan-based inulins and oligofructoses (Roberfroid, 1998) which are currently manufactured in Belgium, France and the Netherlands from chicory roots. Among other plants rich in inulins, Jerusalem artichoke (Helianthus tuberosus L.) stands out as an interesting candidate for the industrial production as its tubers accumulate similar levels of inulin (16–20% of fresh tuber) as chicory roots (Franck, 2000) and could be cultivated at a low cost with low input of fertilisers on any type of soil and cool climatic conditions (Parameswaran, 1994). However, the use of Jerusalem artichoke (JA) tubers for inulin extraction is less well known as they are commonly eaten as vegetable. Our research group has developed protocols for extraction and clarification of inulin from JA tubers (Paseephol et al., 2007) and the aim of this study was to evaluate the prebiotic power of Jerusalem artichoke inulin (JAI) to support the growth and survival of probiotic bacteria in fermented dairy products. Consequently, the objectives of this study were to: (1) Examine the growth of three probiotic strains in the presence of JAI in basal media and select the probiotic strain that shows preference to JAI for subsequent development of synbiotic yoghurt. (2) Determine the effects of inulin powder addition (JAI and chicory inulins) on the acidifying activities and survival of selected probiotic (LC-01) during 28 days of yoghurt shelf life at 4 C.

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2.

Materials and methods

2.1.

Reconstituted skim milks (RSM) and starter cultures

Low-heat skim milk powder (LHSMP, 0.9% milk fat, 34% protein) (Bonlac Foods Ltd., 636 St. Kilda Rd., Melbourne, Vic., Australia) was reconstituted with distilled water to 12% and 16% solids content for yoghurt making. Freeze-dried starter cultures of L. acidophilus LA-5, L. casei LC-01, B. bifidum BB-12 and YC-380 [Yoflex, a 50:50 (dry base) mixture of S. thermophilus (ST) and L. delbrueckii subsp. bulgaricus (LB)] were obtained from Chr. Hansen Pty. Ltd. (49 Barry St., Bayswater, Vic., Australia) and were kept at 22 C until required for yoghurt preparation as direct vat set (DVS).

2.2.

Commercial inulin products

The commercial chicory inulin powders, namely mediumchain inulin (Raftiline GR with an average chain length of 10) and short-chain inulin or oligofructose (OF) (Raftilose P95 with an average chain length of 4) were supplied by Mandurah Australia Pty. Ltd. (Dandenong, Vic., Australia).

2.3.

Jerusalem artichoke inulin (JAI)

The JAI was extracted from the JA tubers using hydrothermal extraction, clarification and concentration process according to Paseephol et al. (2007). The concentrate (P12 B) was then subjected to cold fractionation by storing at 24 C and thawing at 4 C the following day. The insoluble inulin fractions appearing as a dense white substance at the bottom of the containers were recovered by centrifugation at 2700 g and 4 C for 10 min, re-dissolved by warming in a water bath and spray-dried (a Niro laboratory spray drier with a vane type rotary atomiser, Soborg, Denmark) with an inlet temperature of 120 C, outlet temperature of 80 C and air pressure of 5 kg/cm2. The physico-chemical characteristics of experimentally-prepared JAI are compared with two commercial chicory inulin powders in Table 1.

2.4.

Determination of the prebiotic effect of JAI

The growth promoting ability of spray-dried JAI was evaluated on selected probiotics (LA-5, LC-01, BB-12) in basal media in comparison with two commercial chicory inulin powders, all added at 4% level. Control batches contained no prebiotics. The working cultures of LC-01, BB-12 and LA-5 and carbohydrate-free MRS broth were prepared according to Paseephol et al. (2008). To maintain uniform growth conditions throughout the experiments, the sterilised medium was made in large batch and divided into nine pre-sterilised universal bottles (80 ml each). Four grams of each inulin powder were dissolved in 20 ml distilled water and after filter sterilisation the filtrate was added to cooled sterile medium bottle containing 80 ml growth medium to achieve a final inulin concentration of 4% (w/v). Non-supplemented medium (100 ml) was used as the control. One milliliter of each of the probiotic working culture was aseptically transferred into each media bottle and incubated at 37 C for 16 h under aerobic condition for LA-5,

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Table 1 – Physico-chemical properties of inulin powders studied. Raftilose P95a

Properties Structure Average chain length Dry matter (%) Inulin/OF (% d.m.) Sugars (% d.m.) Ash (% d.m.) pH

Raftiline GRa

GFn + Fn 4 P95 95 5 <0.2 5–7

JAI

GFn 12 P95 92 8 <0.2 5–7

GFn + Fn 9 P95 675 615 5 5–7

G, glucose; F, fructose; d.m., dry matter. a Data obtained from Franck (2000).

and an anaerobic atmosphere (Gas-pack, Anaerogen, Oxoid, Basingstocke, UK) for BB-12 and LC-01. The turbidity of the growth media was examined at the end of incubation period by measuring their optical density (OD). The incubated bottles were vortexed for 20 s, and the homogenised media were transferred into optical cuvettes for absorbance readings at 600 nm (n = 3) against the nonsupplemented, non-cultured basal medium (Kneifel et al., 2000).

2.5. Effects of inulin addition on the survival of probiotic bacteria in yoghurt during refrigerated storage The effectiveness of JAI for improving the viability of probiotic and yoghurt cultures was assessed in comparison to chicory inulin powders over 28 days of cold storage (4 C). Three experimental yoghurts, labelled as JAY, INY and OFY were made with 12% RSMs supplemented with 4% of JAI, chicory inulin (Raftiline GR) and oligofructose (Raftilose P95), respectively. Control non-fat yoghurt (16% total solids), labelled as NFCY, was also prepared. Batch coding and the list of all ingredients used are shown in Table 2. The inulin-supplemented RSMs were heat-treated at 90 C for 10 min prior to cooling to 37 C and inoculation with 0.01% each of freeze-dried LC-01 and YC-380 as recommended by the culture manufacturer. After slow agitation for 10–15 min to distribute the culture evenly, the inoculated milk samples were aseptically transferred into 100 ml plastic containers, tightly sealed, incubated at 37 C overnight and transferred to fridge at 4 C the following day. All trials were replicated three times. The sampling schedule for testing was immedi-

ately after the addition of starter culture into milk (day 0), after overnight incubation (day 1), 24 h-post fermentation (day 2) and at weekly intervals (days 7, 14, 21 and 28). The analyses were performed on triplicate samples in triplicate for titratable acidity and pH (Sections 2.6.1 and 2.6.2), and in duplicate for bacterial counts (Section 2.6.3).

2.6.

Analytical determinations of set yoghurt

2.6.1.

Determination of titratable acidity (TA)

Titratable acidity (as % lactic acid) of yoghurt was determined in triplicate according the AOAC titration method 947.05 using 0.1 M NaOH (AOAC, 2000).

2.6.2.

Determination of pH

The pH of yoghurt was measured using a bench-top pH-meter (model 520A, Orion Research Inc., Boston, MA, USA) which was previously calibrated with pH 7.0 and 4.0 standard buffers. All analyses were carried out in duplicate at 20 C.

2.6.3.

Enumeration of probiotic and starter cultures

Ten gram samples of each yoghurt batch were diluted with 90 ml of 0.1% sterile buffered peptone water (Oxoid Ltd., Hampshire, UK) and placed in stomacher for 2 min. Tenfold serial dilutions of 102–109 were then prepared in 9 ml of 0.1% sterile peptone water and 1 ml of the three highest dilutions was pour-plated in duplicate. Counts of LC-01 were enumerated on LC agar under anaerobic condition (Gas-pack, Anaerogen, Oxoid, Basingstocke, UK) at 27 C for 72–96 h. Counts of ST were enumerated on M17 agar (Oxoid Ltd., Hampshire, UK) supplemented with 5% filter-sterilised lac-

Table 2 – Yoghurt production protocol. Code

NFCY JAY OFY INY

Supplement

– JAI Raftilose P95 Raftiline GR

Ingredients (%) LHSMP

Prebiotic

Distilled water

16 12 12 12

0 4 4 4

84 84 84 84

Protein content (%) 5.4 4.1 4.1 4.1

LHSMP, low-heat skim milk powder. NFCY, non-fat control yoghurt; JAY, yoghurt supplemented with 4% JAI; OFY, yoghurt supplemented with 4% oligofructose (Raftilose P95); INY, yoghurt supplemented with 4% chicory inulin (Raftiline GR).

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2.5

LA-5 2.0

OD at 600 nm

tose solution after aerobic incubation at 37 C for 24 h whereas counts of LB were determined on MRS-pH modified agar (pH 5.2) after anaerobic incubation at 43 C for 72 h (Ravula & Shah, 1998). Under these conditions, ST appeared as lenticular colonies with a diameter of 1–2 mm, LB formed lenticular star-shaped colonies 1–3 mm in diameter and LC01 formed smooth white disc colonies measuring 1–2 mm in diameter. The numbers of colony forming units (CFU) on plates containing 25–250 colonies were calculated per gram of sample and the viability of each culture in different samples was calculated as follows (Bruno et al., 2002):

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1.5

1.0

a b

ab

ab

0.5

% Viability ¼ ðCFU=g after 28 days of storage=initial CFU=gÞ  100

0.0

Statistical analysis

All results were statistically analysed using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). The Duncan’s Multiple Range test (DMRT) was applied for mean comparison when one-way analysis of variance (ANOVA) showed significant differences at the 95% confidence level.

3.

Results and discussion

2.5

LC-01

b

b

1.5 1.0 0.5

Two separate experiments were conducted to evaluate the prebiotic power of the JAI and to determine its possible application in dairy products.

c

0.0 2.5

Prebiotic effect of JAI

Fig. 1 shows the capability of utilising various inulin powders by three probiotic strains, expressed as the OD at 600 nm. Overall, the strain of LC-01 could utilise inulin powders the best, followed by BB-12. On the other hand, the growth of LA-5 was minimal in the presence of all inulin powders, showing ODs at 600 nm of ca. 0.7–0.8 compared with ca. 0.9– 2.2 by LC-01 and 1.1–1.6 by BB-12. The growth of LC-01 was highest in broths containing oligofructose (Raftilose P95) while the JAI stimulated the growth of LC-01 to a similar level as the medium-chain inulin (Raftiline GR). Although several studies have reported that the majority of B. bifidum strains were not able to grow on inulins (Hidaka et al., 1986; Bielecka et al., 2002), BB-12 showed efficiency in utilising JAI and chicory inulin powders, especially the OF. This inconsistency may be explained by the differences in the strains used. According to Voragen (1998), variations in chemical structure of saccharides (linear or branched), degree of polymerisation (DP), composition of monomer units and water solubility affect their utilisation by micro-organisms. In the current study, Raftilose P95 was the best utilised material by all three probiotic organisms because of its short chain length, unbranched nature and high water solubility, while the growth of probiotic strains in the presence of higher DP inulins was poor. These findings are in good agreement with Roberfroid et al. (1998) who concluded that short-chain inulin (DP < 10) was the most fermentable substrate, being fermented twice faster than longer-chain inulin. In addition to the DP of inulin chains, the utilisation of inulin by probiotic organisms

BB-12

2.0

OD at 600 nm

3.1.

a

2.0

OD at 600nm

2.7.

a

ab b

1.5 1.0

c 0.5 0.0 CTRL

P95

GR

JAI

Fig. 1 – Growth rates of LA-5, LC-01 and BB-12 in media containing three different inulin powders. Results are mean ± SD of three replicates. Bar followed by different letters differ significantly at P < 0.05 by DMRT. CTRL: control; P95: oligofructose; GR: medium-chain inulin; JAI: Jerusalem artichoke inulin powder.

depends on the purity of the preparations where less purified inulins are fermented more favourably than the highly purified inulins (Biedrzycka & Bielecka, 2004). Raftilose P95 used in this study contained readily available mono- and disaccharides which are better and often not selectively utilised by the bacteria. The ability of JAI in enhancing the growth of the three probiotics was comparable to medium-chain inulin, but not as efficient as oligofructose. Among the three probiotic strains studied, the supplementation of JAI resulted in greater growth rates of LC-01 than BB-12 and LA-5.

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3.2.

Assessment of yoghurt during refrigerated storage

3.2.2.

In this study, synbiotic yoghurt was manufactured to study the effects of inulin powder addition (JAI and chicory inulin powders) on the survival and acidifying activities of probiotic cultures during fermentation and refrigerated storage at 4 C. All yoghurts were inoculated with mixed cultures (1:1, w/w) of LC-01 and traditional yoghurt starters (LB and ST) and incubated overnight at 37 C. The probiotic LC-01 was elected on the basis of the earlier experiment which found maximum activity of this strain in the presence of JAI substrate. The use of yoghurt cultures was necessary to achieve fast development of acidity and yoghurt with desirable rheological characteristics (Tamime et al., 2005).

3.2.1.

Changes in pH and TA

Fig. 2 shows variations in pH and acidity of experimental and control yoghurts during refrigerated storage for up to 28 days. The addition of inulin powders regardless of the type used did not affect the initial pH (6.6–6.7) and TA (0.20–0.27%) of yoghurt milks. There were no significant differences (P > 0.05) in the pH and TA values of supplemented and non-supplemented control yoghurt (NFCY) throughout the storage period. A similar trend was also reported by Guven et al. (2005) and Zhu (2004). On day 1, the acid production in JAI-supplemented yoghurt (JAY, pH 4.2, TA 1.02%) was higher, but not statistically significantly differently compared to those of chicory oligofructose (OFY, pH 4.4, TA 0.86%) and inulin-containing yoghurts (INY, pH 4.5, TA 0.79%). All yoghurts containing prebiotic substrates had a stable pH and acidity over time, showing maximum of 0.2 pH unit drops and a 0.28% increase in TA at the end of storage. On day 28, the pH of the yoghurts averaged from 4.3 (for INY) to 4.1 (for OFY and JAY) while the acidity of all samples varied from 1.05% (for INY) to 1.20% (for NFCY). The low level of post-acidification in these yoghurts could be attributed to the type of probiotic and yoghurt starters used.

7.0

1.3

6.5

1.1

6.0

pH

0.7 5.0

%TA

0.9 5.5

0.5 4.5 0.3

4.0 3.5

0.1 0

7

14 Period (day)

21

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28

Fig. 2 – Changes in pH (dotted lines) and TA (solid lines) of four yoghurts during fermentation and storage at 4 C. Triangle: INY; Cross: OFY; Diamond: JAY; Square: NFCY.

315

Changes in the counts of probiotic and yoghurt bacteria

A variation in viable counts of LC-01, ST and LB in yoghurts with and without inulin supplementation during fermentation and over the storage period of 28 days is presented in Table 3. Overall, the retention of viability of STwas better than those of LB and LC-01. Compared to the control, the addition of inulin powders did not influence the survival of ST and LB, but significantly improved the viability of LC-01. Among the three inulin powders tested, the best retention of LC-01 numbers was observed with chicory inulin (Raftiline GR, 7.4 log CFU/g) followed by oligofructose (Raftilose P95, 7.3 log CFU/g) and JAI (7.1 log CFU/g). The initial counts of ST in all inoculated milks ranged from 5.3 to 5.5 log CFU/ml that increased by ca. 3 log cycles after overnight incubation. No significant difference (P > 0.05) in the counts of ST was observed between non-supplemented and supplemented yoghurt batches throughout the storage period. This is supported by Kaplan and Hutkins (2000) who reported that several strains of L. casei and L. acidophilus were able to ferment fructo-oligosaccharides well, but not most of the LB and ST strains. The experimental and control yoghurts showed ability in sustaining high numbers of ST up to 14 days of storage (P > 0.05) and only a marginal decline occurred in the following 14 days. After 28 days, all yoghurts contained >8.0 log CFU/g of ST, decreasing from the initial counts by only 2.7–4.2%. This reflected the high stability of ST in the products. These observations were consistent with the findings of Akalin et al. (2004), Dave and Shah (1997), Medina and Jordano (1994) and Ozer et al. (2005) who reported higher stability of ST than LB and bifidobacteria in probiotic yoghurts during storage time. The initial counts of LB After overnight incubation in all yoghurts were comparable to those of LC-01 and ST (ca. 8.1 log CFU/g). Supplementation with inulin powders did not help the viability of LB as their numbers in all supplemented yoghurts dropped by ca. 1 log after 14 day of storage, similar to those in the controls. An steady decline in the numbers of LB was observed until the end of the storage period wherein the final counts were ca. 6.2–6.4 log CFU/g (76–78% of the initial counts). Ozer et al. (2005) also reported the decline of viable counts of LB by 2.5–4.2 times in inulin-supplemented yoghurts during 14 days of storage. Several workers have reported that low numbers of LB would help the survival of probiotic organisms due to reduced risks of post-acidification by LB (Holcomb & Frank, 1991; Shah, 1995). The addition of inulin powders was helpful in improving the growth of LC-01 during fermentation and their survival during storage time. The results of this study showed a higher initial count of LC-01 (P 6 0.05) in all inulin-supplemented yoghurts (ca. 8.3 log CFU/g) compared with the control without inulin (ca. 8.0 log CFU/g). These results are consistent with the findings of Aryana and McGrew (2007) who reported a marked increase in L. casei counts with the addition of inulin powders. During storage, the counts of LC-01 for all trials declined with time and became significant on day 7 (P < 0.05), but the rate of decline was slower in the presence of inulin powders. In all inulin-supplemented yoghurts, viable counts of LC-01 remained above 8.0 log CFU/g up to 14 days of storage which gradually dropped to 7.0 log CFU/g by the 28th day of

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Table 3 – The viable counts (log CFU/g) of LC-01, ST and LB in yoghurts with and without inulin addition during fermentation and storage at 4 C. Culture LC-01

ST

LB

Period

NFCY

JAY e

OFY e

INY e

0 1 2 7 14 21 28

5.26 ± 0.08 8.01 ± 0.22a 7.98 ± 0.12a 7.86 ± 0.46a 7.52 ± 0.46b 7.11 ± 0.13c 6.06 ± 0.31d

5.31 ± 0.09 8.26 ± 0.13a 8.20 ± 0.11ab 8.21 ± 0.14ab 8.10 ± 0.16b 7.42 ± 0.16c 7.10 ± 0.15d

5.28 ± 0.08 8.34 ± 0.14a 8.34 ± 0.18a 8.20 ± 0.19ab 8.15 ± 0.16b 7.54 ± 0.14c 7.30 ± 0.20d

5.44 ± 0.04e 8.21 ± 0.15ab 8.28 ± 0.07a 8.20 ± 0.13ab 8.14 ± 0.12b 7.89 ± 0.06c 7.37 ± 0.07d

% Viability

75.66C

85.96B

87.53AB

89.75A

0 1 2 7 14 21 28

5.32 ± 0.08d 8.29 ± 0.09a 8.27 ± 0.08ab 8.26 ± 0.09ab 8.14 ± 0.19abc 8.09 ± 0.15bc 8.02 ± 0.30c

5.46 ± 0.05d 8.36 ± 0.14a 8.35 ± 0.07a 8.26 ± 0.16ab 8.21 ± 0.09ab 8.12 ± 0.11bc 8.01 ± 0.33c

5.42 ± 0.11c 8.38 ± 0.12a 8.31 ± 0.11a 8.34 ± 0.10a 8.28 ± 0.16a 8.12 ± 0.16b 8.13 ± 0.14b

5.46 ± 0.12c 8.29 ± 0.16a 8.29 ± 0.11a 8.29 ± 0.09a 8.20 ± 0.21ab 8.13 ± 0.14ab 8.07 ± 0.17b

% Viability

96.74A

95.81A

97.02A

97.35A

0 1 2 7 14 21 28

5.29 ± 0.12e 8.18 ± 0.10a 8.16 ± 0.11a 7.67 ± 0.19b 7.00 ± 0.16c 6.33 ± 0.23d 6.20 ± 0.13d

5.36 ± 0.16f 8.13 ± 0.35a 8.13 ± 0.21a 7.71 ± 0.24b 7.03 ± 0.15c 6.49 ± 0.08d 6.20 ± 0.16e

5.27 ± 0.08e 8.15 ± 0.16a 8.13 ± 0.20a 7.72 ± 0.13b 7.02 ± 0.16c 6.36 ± 0.34d 6.23 ± 0.28d

5.44 ± 0.12f 8.16 ± 0.11a 8.16 ± 0.16a 7.84 ± 0.16b 7.02 ± 0.14c 6.64 ± 0.09d 6.39 ± 0.09e

% Viability

75.79A

76.26A

76.44A

78.31A

Data are means ± SD of three experiments, and each experiment was examined in duplicate. % Viability = (CFU/g after 4 week storage/initial CFU/g) · 100. NFCY, non-fat control yoghurt; JAY, yoghurt supplemented with 4% JAI; OFY, yoghurt supplemented with 4% oligofructose (Raftilose P95); INY, yoghurt supplemented with 4% chicory inulin (Raftiline GR). a–f Means in the same column with different letters differ significantly at P < 0.05 by DMRT. A–C Means in the same row with different letter differ significantly at P < 0.05 by DMRT.

storage. As shown in Table 3, the highest viability of LC-01 on day 28 was found in INY, followed by OFY, recorded at 90% and 88% viability, respectively, whereas the lowest viability was observed with NFCY at 6.1 log CFU/g (76%). The JAI had a comparable effect on the viability retention of LC-01 to oligofructose (Raftilose P95), with average values of 86%, but was slightly less effective than that of inulin (Raftiline GR). These results seem to corroborate the findings of Aryana et al. (2007) and Lankaputhra et al. (1996) who found higher counts of probiotic bacteria in yoghurts with medium- and long-chain inulins than those with OF at the end of storage. This phenomenon was ascribed to the larger amounts of acid developed in yoghurts with OF. The utilisation of inulin powders by various probiotic organisms has been reported earlier by Akalin et al. (2004), Bruno et al. (2002), Shin et al. (2000) and Varga et al. (2003) who found a significant improvement in the viability retention of bifidobacteria in yoghurts containing prebiotics (inulin/OF) during storage. Similarly, Aryana et al. (2007), Capela et al. (2006), Desai et al. (2004) and Donkor et al. (2007) observed that chicory-based inulins were the favoured carbon source for Lactobacillus strains, hence increasing the growth performance and sustaining the viability during storage. However, some other publications by Bozanic et al. (2002)

and Ozer et al. (2005) reported that ‘‘inulin did not support the growth and survival of L. acidophilus in fermented bovine milk and acidophilus–bifidus yoghurts’’. The inconsistencies reported here could be attributed mainly to strain-specific response of probiotics to prebiotic supplementation. The mechanism by which inulin improves the viability of the probiotic organisms during cold storage is still unclear. The two possible mechanisms proposed so far state that inulins provide additional nutrients for promoting culture growth (Makras et al., 2005) and that they protect probiotic cells from acid injury (Desai et al., 2004).

4.

Conclusions

Studies in modified MRS broths indicated that the 4% supplementation level of JAI resulted in greater growth rates of LC01 than BB-12 and LA-5. Compared to commercial chicory inulin products, the ability of JAI in improving the growth of probiotic bacteria was comparable to medium-chain inulin, but not as efficient as oligofructose. The addition of inulin powders improved the survival of probiotic LC-01 during yoghurt shelf life at 4 C, showing ca. 1 log cycle higher number than non-supplemented yoghurt at the end of day 28. The JAI had the effect on sustaining the viability of LC-01 comparable

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to that of oligofructose whereas the most effective inulin powder was medium-chain chicory inulin. There was no improvement in the growth and survival of yoghurt bacteria in the presence of inulin. The numbers of ST in the experimental and control yoghurts were stable with >8 log CFU/g throughout storage time while the numbers of LB dropped below 8 log CFU/g from week 1 onwards. The post-acidification was found minimal in all yoghurts during refrigerated storage. The initial pH prior to storage ranged from 4.2 to 4.5 that dropped to 4.1–4.3 after 28 days.

Acknowledgements The authors would like to thank Mandurah Australia Pty. Ltd., Australia for the supply of chicory inulin products and RMIT Food Science technical staff for their assistance.

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