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Effects of cross-linked inulin with different polymerisation degrees on physicochemical and sensory properties of set-style yoghurt
Accepted Manuscript Effects of cross-linked inulin with different polymerisation degrees on physicochemical and sensory properties of set-style yoghurt Yao Li, Kinyoro Ibrahim Shabani, Xiaoli Qin, Rong Yang, Xuedong Jin, Xiaohan Ma, Xiong Liu PII:
S0958-6946(19)30052-4
DOI:
https://doi.org/10.1016/j.idairyj.2019.02.009
Reference:
INDA 4464
To appear in:
International Dairy Journal
Received Date: 31 October 2018 Revised Date:
17 February 2019
Accepted Date: 19 February 2019
Please cite this article as: Li, Y., Shabani, K.I., Qin, X., Yang, R., Jin, X., Ma, X., Liu, X., Effects of crosslinked inulin with different polymerisation degrees on physicochemical and sensory properties of setstyle yoghurt, International Dairy Journal, https://doi.org/10.1016/j.idairyj.2019.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of cross-linked inulin with different polymerisation degrees on
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physicochemical and sensory properties of set-style yoghurt
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Yao Li, Kinyoro Ibrahim Shabani, Xiaoli Qin, Rong Yang, Xuedong Jin, Xiaohan Ma,
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Xiong Liu*
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College of Food Science, Southwest University, 400715 Chongqing, China
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*Corresponding author. Tel.: +86 23 68250375
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E-mail address: [email protected] (X. Liu)
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ABSTRACT
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This work investigated effects of cross-linked inulin with different degrees of
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polymerisation (DP, average = 7 and 15) on physicochemical and sensory properties
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of set-style yoghurt. Compared with set-style yoghurt made with native inulin
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(average DP = 4 and 11), yoghurts with cross-linked inulin had higher acidity and
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lower syneresis values, with a shelf-life of 14 days. Supplementation of cross-linked
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inulin with higher DP resulted in enhanced firmness and adhesiveness of yoghurts. In
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addition, bacterial counts showed that yoghurts with cross-linked inulin exhibited
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longer retention of Streptococcus thermophilus and Lactobacillus delbrueckii ssp.
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bulgaricus cell viability than that with native inulin. Sensory evaluation indicated that
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yoghurt with cross-linked long-chain inulin received higher scores for overall
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acceptability than other samples. However, different types of inulin did not
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significantly affect odour and colour of set-style yoghurt. Consequently, cross-linked
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inulin prepared can be exploited as a prebiotic to prolong shelf-life of yoghurt.
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1.
Introduction
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Inulin is a polysaccharide composed of numerous fructose units and a terminal glucose unit. It can be exacted from various plants, such as onions, garlics, bananas
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and chicory roots. Native inulin has varying degrees of polymerisation (DP) ranging
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from 2 to 60 monosaccharide units, which depends on the source, harvest time and
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production process (Glibowski & Zielińska, 2015). Inulin preparations with different
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DP have varying physicochemical properties. Generally, long-chain inulin can act as a
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fat replacer or texture modifier due to its poor solubility and good viscosity stability,
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while short-chain inulin can contribute to improved mouthfeel due to good solubility
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and mild sweetness (Glibowski & Rybak, 2016). As a non-digestible dietary fibre and
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attractive prebiotic, inulin exhibits many beneficial physiological functions, such as
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antioxidant activity, ability to improve colonic microbiota, promoting mineral
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absorption, and regulating blood glucose and lipids (Balthazar et al., 2016).
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Based on nutritional and physicochemical properties, inulin has been widely
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used in an increasing number of food applications. Inulin has proven to be a
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promising fat substitute and to improve the functional potential in sheep milk ice
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cream formulation (Balthazar et al., 2017, 2018). Inulin can also be added for
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stabilisation in the formulation of whey beverage (Guimaraes et al., 2018a) and may
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enhance the viability of Lactobacillus in probiotic yoghurt during storage (Canbulat &
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Ozcan, 2015).
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However, native inulin (a mixture of short-chain and long-chain inulin) has usually been employed without classification in most recent studies, and the lack of
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certainty of the DP of inulin leads to variable effects on food products since inulin
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with different DP has different effects. Luo et al. (2017) reported that inulin with
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lower DP displayed stronger suppression of the retrogradation of wheat starch
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compared with higher DP inulin. Guimaraes et al. (2018b) showed that inulin with
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higher DP had better efficiency for beverage stabilisation. Nevertheless, there is still a
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lack of information about the effect of inulin with different DP on properties of other
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food products. Moreover, short-chain inulin may induce rapid renal excretion due to
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its low molecular weight. Cross-linking of inulin to increase its molecular weight is a
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strategy to avoid this disadvantage (Li et al. 2010). However, there is still no reported
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research exploring the influence of cross-linked inulin with different polymerisation
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degrees on quality attributes of food products, especially on such a popular dairy food
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as set-style yoghurt.
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Therefore, the present work was designed to prepare various set-style yoghurts containing native inulin or cross-linked inulin of different DP and aimed to evaluate
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the different effects of native inulin and cross-linked inulin on physiochemical
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properties and sensory properties of yoghurt, such as water retention, acidity,
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adhesiveness, firmness, colour, taste, and bacterial counts.
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2.
Materials and methods 4
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2.1.
Materials
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Native inulins (average DP < 10) were supplied by Xi’an Ruiling Technology Co., Ltd. (Xi’an, China). Four types of inulin with different DP, i.e., native inulin S-In
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(average DP = 4), native inulin L-In (average DP = 11), cross-linked inulin CS-In
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(average DP = 7), and cross-linked inulin CL-In (average DP = 15), were prepared in
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our laboratory by the method described in Section 2.2. Commercial yoghurt starter
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culture (YFL-702, Chr. Hansen, Denmark) containing Streptococcus thermophilus and
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Lactobacillus delbrueckii ssp. bulgaricus was used. Milk powder was obtained from
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Nestle Co., Ltd. (Switzerland). Peptone water was purchased from Qingdao Jisskang
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biochemical Co., Ltd. (China). Anhydrous ethanol, sodium hexametaphosphate (SH),
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hydrochloric acid, phenolphthalein, sodium hydroxide, glutaraldehyde, potassium
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dihydrogen phosphate, tertiary butyl alcohol were purchased from Kelong Chemical
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Reagent Factory (Chengdu, China). All reagents were of analytical grade.
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2.2.
Preparation of cross-linked inulin
Native inulin (16 g) was added to 80 mL distilled water at room temperature
and gently stirred until dissolved. Exactly 20 mL of anhydrous ethanol was added into the solution. The mixture solution was stored at 4 °C for 24 h, after which sediment 5
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sediment was lyophilised with vacuum freeze-drying equipment (Sihuan LGJ-25C,
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Beijing, China) at 5 Pa and –50 °C for 12 h; the product obtained was long-chain
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inulin (L-In) in native inulin. The liquid supernatant was condensed by rotary
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evaporation (Rongsheng RE-52CS, Shanghai, China) followed by lyophilising for 24
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h (5 Pa, –50 °C); the product obtained was short-chain inulin (S-In). Cross-linked
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inulin was prepared by a method similar to that of starch modification (Ren, Jiang,
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Wang, Zhou, & Tong, 2012). Typically, L-In (10 g) was dispersed in water (200 mL)
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with constant stirring for 1 h at 25 °C and then 1.1 g of cross-linker SH was added
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followed by the addition of Na2CO3 (2 M) to attain pH 10. Then the
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pre-polymerisation solution was polymerised with continuous stirring at 45 °C for 3.5
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h. Upon completion of the reaction, the pH of the suspension was adjusted to 7 with 2
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M
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centrifugation and washing with anhydrous ethanol. Finally, after lyophilising,
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cross-linked long-chain inulin (CL-In) was obtained. Cross-linked short-chain inulin
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(CS-In) was prepared in the same manner but with the S-In in place of L-In. The
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molecular structural formula of cross-linked inulin is illustrated in Fig. 1 (Li, Ma &
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Liu, 2018).
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HCl. The cross-linked inulin was then precipitated with 95% ethanol, followed by
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2.3.
Preparation of yoghurt
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Non-supplemented (inulin-free) control yoghurt was prepared by dispersing 16.0% (w/w) milk powder and 8% sugar in deionised water. Inulin-supplemented
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yoghurts were made from 16.0% (w/w) milk powder, 6% sugar and 2% corresponding
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inulin (respectively, S-In, L-In, CS-In, and CL-In) and deionised water. All
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ingredients were weighed in a flask and mixed with continuous agitation (Sile
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HD2004W, Shanghai, China) at 60 °C until completely dissolved. The reconstituted
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milk mixtures were homogenised (20 MPa, 60 °C) in a homogeniser (Nuoni
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GJJ-0.06/40, Zhejiang, China) and then flasks with samples were heated in a water
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bath at 95 °C for 15 min and then rapidly cooled in chilled water to 45 °C. After that,
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all the mixes were each inoculated with 1 g L-1 of yoghurt culture YFL-702, followed
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by incubation at 42 °C for 5 h. When acidity reached 0.8% lactic acid, the yoghurt
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was cooled to 4 °C.
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2.4.
Physicochemical determinations
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Acidity, expressed as g of lactic acid per 100 g of yoghurt, was determined on
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days 1, 4, 7, 11 and 14 according to the titration method of the Association of Official
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Analytical Chemists (AOAC, 2002).
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To measure syneresis (g per 100 g), a batch of prepared set-style yoghurt (25 g)
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was weighed in a centrifuge tube and then stored at 4 °C for 1, 4, 7, 11, and 14 days.
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Each sample was centrifuged at 3000 × g (Centrifuge 5810, Eppendorf, Germany) at 7
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fine mesh screen placed on top of a funnel. The clear supernatant was poured off, then
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weighed and expressed as percentage weight relative to the original weight of yoghurt
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(Srisuvor, Chinprahast, Prakitchaiwattana, & Subhimaros, 2013). All analyses were
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carried out in triplicate.
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Textural characteristics such as hardness, cohesiveness, and adhesiveness were evaluated using a texture analyser (CT-3; Brookfield, USA) equipped with a
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TA4/1000 cylindrical probe (38.1 mm diameter). The yoghurt samples prepared were
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stored at 4 °C for 24 h before analysis. The texture analysis circumstances were as
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follows: penetration depth, 30 mm; force, 5 g; probe speed during penetration, 1 mm
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s-1 (Tiwari, Sharma, Kumar, & Kaur, 2015). The samples, kept in original containers
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with a diameter of 70 mm, were penetrated at ambient temperature (10 ± 1 °C). Two
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cycles were applied; the values for texture attributes were obtained from the resulting
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curve using the Texture Pro CT equipment software. The following parameters were
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quantified: firmness as defined as the maximal peak value recorded in the first
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immersion into the sample; cohesiveness defined as the ratio of positive force area
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during the second immersion cycle to that of the first immersion cycle; and
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adhesiveness, or the negative area for the first cycle, representing the work necessary
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to pull the probe away from the sample.
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The colours of set-style yoghurts stored at 4 °C for 24 h were measured with a
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colorimeter (Minolta, Spectrophotometer CM-3500d, Japan). The colorimeter used L* 8
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(lightness), a* (redness) and b* (yellowness) scales, and operating conditions were
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illuminant D65 and 2° sample-observer. Colour was measured in triplicate for each
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sample (Akbari, Eskandari, Niakosari, & Bedeltavana, 2016).
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2.5.
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Determination of bacterial counts
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A representative sample of yoghurt (1 g) was suspended in 9 mL of sterile 0.1% (w/v) peptone water (Jisskang, Qingdao, China) and subsequently serially diluted to
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10-7. Counts of S. thermophilus (ST) were enumerated using the pour plate technique
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after aerobic incubation at 37 ºC for 72 h on M17 agar (Merck, USA) and
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enumeration of L. delbrueckii ssp. bulgaricus (LB) was performed using MRS agar
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(Merck, USA) incubated at 37 °C for 72 h under anaerobic conditions, according to
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the technique for yoghurt enumeration of characteristic microorganism colony count
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described by International Organisation for Standardisation (ISO, 2003).
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2.6.
Sensory evaluation
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Sensory evaluations were carried out 3 days after production and by a group of
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20 panellists (10 females and 10 males, aged between 17 and 38 years old) who were
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trained in sensory analysis of dairy products. Panellists evaluated the samples and
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recorded their perceptions by making marks on a ten-score grades as described by 9
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unnatural colour, 10 – no criticism), taste (1 – bad, 10 – no criticism), odour (0 –
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undetectable, 10 – very intensive), texture and consistency (0 – liquid, 10 – very
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thick), and overall acceptability (0 – dislike extremely, 10 – like extremely).
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Definitions for the descriptors describing the colour, taste, odour, texture and
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consistency of set-style yoghurt were developed by panel consensus using reference
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samples. Sample defects and descriptors such as acid, sweet, bitter, acetaldehyde taste,
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milky, creamy, grainy, off-flavour, ropy and gritty texture, whey syneresis, and
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lumpiness were recorded. Each sample (30 mL) was scored individually, and samples
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were presented to the panellists in random and balanced order in white plastic cups
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coded with three-digit random numbers. Mineral water was provided for mouth
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rinsing in between samples.
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2.7.
Statistical analysis
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All experiments were conducted in triplicate for each sample, and results were
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expressed as mean ± standard deviation. All data were statistically compared between
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groups using one-way analysis of variance. Significant differences among the mean
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values were determined by Duncan’s multiple range test with p < 0.05.
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3.
Results and discussion 10
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3.1.
Analysis of acidity
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The titratable acidity values of the stored set-style yoghurt are presented in
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Table 1. An increase in titratable acidity was observed over storage time, due to more
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lactic acid production and the higher acidity in the yoghurt caused by continuous
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fermentation and stimulated growth of lactic bacteria, which often results in greater
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contraction of the casein micelle matrix and increased expulsion of whey (Achanta,
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Aryana, & Boeneke, 2007). Thus, the shelf-life of set-style yoghurt is often limited by
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excessive acidification during storage (Akalin, Gönç, Unal, & Fenderya, 2007).
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Compared with yoghurt containing inulin, the control yoghurt had the lowest acidity value, which agrees well with that reported by Aryana et al. (2007) and Guven
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et al. (2005). According to research of Perrin et al. (2002), the acidity of yoghurt
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containing lower DP inulin was higher than that of others, due to the faster
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consumption by the probiotic bacteria which could increase lactate production.
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However, data in Table 1 are not consistent with this trend, as these results showed
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that set-style yoghurt containing 2% cross-linked inulin (CS-In or CL-In) had
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significantly higher acidity than that with corresponding native inulin (S-In or L-In),
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while the CS-In yoghurt had a higher acidity than the CL-In yoghurt. Consequently,
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acidity values of yoghurts were mainly influenced by the introduction of acid
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phosphate groups and DP of inulin.
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According to the result of a study of acid stability of inulin (Li et al., 2018), breakdown and hydrolysis would occur for the cross-linked inulin in acidic
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environment (pH <6) and inulin with higher DP was more difficult to transform into
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reducing sugar. The pH value of yoghurt is about 4, so cross-linked inulin should
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partly break down to reducing sugar and phosphoric acid in this acidic condition, and
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the generated phosphoric acid would further increase the yoghurt acidity.
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3.2.
Analysis of syneresis
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Syneresis value could reflect the water-holding capability of yoghurt product during storage. The syneresis values of yoghurt samples over storage are presented in
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Table 2. Both inulin polymerisation degree and storage time had significant effects on
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syneresis. There was a steady increase in syneresis with storage time for all yoghurt
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samples, including the control, which may be explained in terms of the increase in
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acidity during storage causing the casein micelle matrix to contract and extrude more
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whey (Canbulat & Ozcan, 2015). The extent of syneresis was affected by the addition
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of inulin. Yoghurt samples made with inulin underwent less syneresis than the control,
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which may be explained by the ability of inulin molecules to bind water, preventing
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free movement of water, and to complex with protein aggregates and provide stability
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to the protein network. In fact, many studies have shown decreased syneresis of
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yoghurt following the addition of inulin (Meyer, Bayarri, Tárrega, & Costell, 2011).
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Interestingly, yoghurts made with CL-In had significantly lower syneresis values than those of yoghurt made with other inulins, which suggests that CL-In
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possibly had a better water-holding capacity than L-In, S-In, and S-In, due to the
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higher DP. This result was consistent with that of Aryana and Mcgrew (2007) who
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reported that long-chain prebiotic had a better water-holding capacity than that of
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short-chain prebiotic in yoghurt. A good-quality yoghurt should hold water without
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syneresis. Therefore, the utility of CL-In in this study to act as a food stabiliser could
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contribute to the yoghurt gel structure, preventing fracture and whey-off.
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3.3.
Analysis of textural characteristics
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Table 3 shows the effect of adding different types of inulin at a level of 2% on
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textural parameters of set-style yoghurts. Some significant differences in textural
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parameters were observed among different yoghurt samples. The addition of S-In and
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CS-In, composed of short-chain inulin, decreased the firmness of control yoghurt
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compared to L-In and CL-In with higher DP. Set yoghurt made with cross-linked
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inulin (CS-In and CL-In) had higher firmness than that with corresponding native
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inulin (S-In and L-In), which can be explained by the fact that the viscosity of inulin
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increases with the degree of polymerisation and cross-linked inulin had a higher
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viscosity and water-binding capacity than native inulin, which caused enhanced
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molecular flow resistance and decreased molecular mobility (Akalin, Unal, Dinkci, &
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firmness of yoghurt. Some studies have shown decreased firmness of yoghurt with
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added inulin (Kusuma, Paseephol, & Sherkat, 2009), and the lower firmness of
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inulin-supplemented yoghurts may be attributed to short-chain inulin not generating
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strong viscosity and possibly interfering with the formation of the protein network,
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resulting in softening of yoghurt. Therefore, the DP of added inulin had an important
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influence on firmness of yoghurt, a result consistent with that of Glibowski &
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Kowalska (2012).
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The same observations were made for adhesiveness, which is defined as the work required to overcoming the attraction forces between the surface of yoghurt and
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the probe surface (Chiavaro, Vittadini, & Corradini, 2007). Yoghurt containing inulin
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with higher DP had greater adhesiveness. The presence of CL-In significantly
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increased the work required to remove yoghurt that adheres to the probe. This result is
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in agreement with that of Villegas and Costell (2007), who showed that addition of
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inulin with higher DP resulted in significantly enhanced adhesiveness of whole milk.
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Values of firmness and adhesiveness of yoghurt made with CL-In were higher than
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those of the yoghurts made with other inulin types, which could be attributed to that
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inulin of higher DP generating a higher viscosity and water-binding capacity.
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Cohesiveness ratio, expressed as values between 0 and 1, show the ability for
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rebuilding the structure of the sample. The highest value of cohesiveness was shown
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by control yoghurt, with slightly lower cohesiveness by yoghurt with inulin. A lower 14
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cohesiveness ratio was observed in yoghurt with CL-In, which could be due to inulin
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of higher DP being dispersed within the casein micelles, interfering with protein
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matrix formation (Meyer et al., 2011).
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3.4.
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Counts of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus
Changes in counts of S. thermophilus and L. delbrueckii ssp. bulgaricus over a
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storage period of 14 days are presented in Table 4. Storage time markedly influenced
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the levels of S. thermophilus and L. delbrueckii ssp. bulgaricus in CL-In yoghurt
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throughout 14 days of storage, while statistical differences of counts for other sample
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groups (the control, CS, CS-In and CL) could be observed only between day 1 and
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day 4. This is in agreement with results of Balthazar et al. (2016), who reported
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significant changes occurred in S. thermophilus and L. delbrueckii ssp. bulgaricus
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counts during the storage of yoghurt. Although very few statistical differences were
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observed between samples (control and different inulin types) for each day of storage,
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overall, the counts of S. thermophilus and L. delbrueckii ssp. bulgaricus in yoghurts
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with inulin were slightly higher than that of the control after storage of day 4. The
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result was consistent with Akalin, Fenderya, and Akbulut (2004), who reported higher
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counts of two commercial strains of bifidobacteria in yoghurts containing
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fructo-oligosaccharide compared with yoghurts without this prebiotic. At the
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thermophilus and L. delbrueckii ssp. bulgaricus, varying from 7.46-7.82 log cfu g-1
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for S. thermophilus and 7.58-7.81 log cfu g-1 for L. delbrueckii ssp. bulgaricus. The
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numbers of S. thermophilus and L. delbrueckii ssp. bulgaricus in control, S-In, CS-In
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treatments remained marginally increased up to day 7, but slowly declined over the
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following week, whereas the counts of S. thermophilus and L. delbrueckii ssp.
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bulgaricus in L-In, CL-In yoghurts slightly rose up to day 11 and then decreased. By
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day 14, the populations of S. thermophilus and L. delbrueckii ssp. bulgaricus in all
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yoghurt samples increased compared with the beginning, while the increase in
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yoghurts containing CL-In was most noticeable (1.48 log cfu g-1 for S. thermophilus
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and 1.5 log cfu g-1 for L. delbrueckii ssp. bulgaricus).
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of Lactobacillus acidophilus in yoghurts supplemented with long-chain inulin than
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yoghurts with short-chain inulin at the end of storage. Yoghurts containing S-In and
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CS-In showed slightly higher counts in both S. thermophilus and L. delbrueckii ssp.
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bulgaricus than that of L-In and CL-In on day 1 and day 4, suggesting that
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supplementation with inulin with an increased DP led to a decreased rate of
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consumption of inulin by S. thermophilus and L. delbrueckii ssp. bulgaricus.
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According to Akalin et al. (2007), the prebiotic activity of inulin was dependent on
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DP of inulin and shorter-chain inulin is more commonly used as a prebiotic. However,
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this trend was reversed at subsequent storage up to day 11 and day 14, as the S.
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CL-In were higher than that of S-In, CS-In and the control. This may be explained by
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the high acidity of yoghurts with short-chain inulin, consistent with the findings of
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Lankaputhra, Shah, and Britz (1996) and Cruz et al. (2009) that high yoghurt acidity
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negatively affected the viability of probiotic bacteria. Overall, yoghurts with added
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CL-In exhibited longer retention of S. thermophilus and L. delbrueckii ssp. bulgaricus
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cell viability than yoghurts made with other DP inulin.
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3.5.
Colour characteristics
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The colour values (L*, a*, b*) of different yoghurts are summarised in Table 5. For different yoghurts, a* values varied between –1.69 and –1.17, and b* values varied
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from 7.17 to 11.13 while, overall, both a* and b* values increased with higher inulin
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DP. Addition of inulin gave slightly higher L*, b* and a* values than the control. These
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changes in colour may be associated with inulin molecules interacting with casein
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micelles, which along with the fat globules, are responsible for the diffusion of
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incident light. In contrast, Aryana et al. (2007) reported that addition of 1.5% (w/w)
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inulin in yoghurts did not have any significant effect on L∗, a∗, or b∗ values. The
350
different result can be explained by the higher concentration of casein and fat globules
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in this matrix and higher inulin amount used in this work. However, Balthazar et al.
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(2015) observed slightly higher L*, b* and a* values for ovine milk yoghurt with inulin,
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ACCEPTED MANUSCRIPT in agreement with this work. In addition, acid hydrolysis of inulin can probably occur
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in yoghurt containing inulin due to the presence of lactic acid formed by the starter
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culture and adequate fermentation temperature (42 ± 1 °C), interfering with the colour
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of yoghurts. Schreiner (1950) reported a colorimetric method for the determination of
357
inulin by means of resorcinol, due to the ability of inulin to change the medium colour
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after acid hydrolysis above 40 °C. Although all yoghurt samples appeared white to the
359
naked eye, the instrument picked up slight green and yellow colours.
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3.6.
Sensory evaluation
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Sensory evaluation to complement the physicochemical properties of the yoghurt samples were performed (Table 5). No significant effect of inulin on odour,
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colour and taste was observed. However, significant differences were observed
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between the sensory evaluations regarding the texture and consistency and overall
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acceptability of yoghurt samples. For overall acceptability, the sensory scores of the
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yoghurt containing CL-In and L-In were slightly higher than those of the control
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yoghurt and the yoghurt containing S-In and CS-In. In addition, the panellists deemed
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that S-In and CS-In yoghurts were slightly sweeter than CL-In and CL-In yoghurts,
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which tasted more smooth and creamy, this indicating that taste and texture of yoghurt,
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to a certain extent, was influenced by the presence of inulin. This is in agreement with
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the result of Canbulat and Ozcan (2015), who reported that short-chain inulin has 30%
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in chain length between short-chain inulin and long-chain inulin account for their
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distinctly different functionality attributes, short-chain inulin possesses functional
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qualities similar to sugar or glucose syrup, etc., while long-chain inulin has been used
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successfully to replace fat in dairy products and baked goods.
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Several authors have studied the replacement of sugar and fat with different
types of inulin in food products. Tarrega and Costell (2005) showed that adding inulin
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to fat-free dairy model desserts significantly increased sweetness compared with those
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with no addition. Rodríguez-García, Salvador, and Hernando (2014) reported the good
383
functionality of short-chain inulin as replacers for sugar in low-sugar cakes and
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long-chain inulin as replacers for fat in low-fat cakes. Another interesting observation
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was that of worse texture and consistency scores given to CS-In in comparison with
386
other types of inulin yoghurts. Similar results have been reported in some studies
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concerning yoghurts with inulin. Aryana et al. (2007) reported that inulin chain length
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had insignificant effect on colour and appearance of fat-free yoghurt, but that
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medium-chain and long-chain inulin gave yoghurt better sensory properties. Yi,
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Zhang, Hua, Sun, and Zhang (2010) found that inulin and its hydrolysate could
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partially replace sucrose in yoghurt. Kip, Meyer, and Jellema (2006) observed that
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moderately long-chain inulin could be used successfully to improve the creamy
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mouthfeel of low-fat yoghurt, whereas Guven et al. (2005) found that excessive
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addition of inulin in fat-free yoghurt negatively influenced some physical properties
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of yoghurt, i.e., whey separation, consistency and organoleptic scores.
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4.
Conclusions
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Compared with set-style yoghurt made with native inulin (S-In and L-In), the
yoghurt samples supplemented with cross-linked inulin (CS-In and CL-In) had higher
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acidity and lower syneresis values during the observed shelf-life of 14 days,
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indicating that inulin with higher DP had better water-holding capacity.
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Supplementation of cross-linked inulin with higher DP resulted in enhanced firmness and adhesiveness of yoghurt. In addition, bacterial counts indicated that the
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addition of cross-linked inulin as a prebiotic to the yoghurt led to longer retention of S.
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thermophilus and L. delbrueckii ssp. bulgaricus cell viability during storage.
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Moreover, sensory evaluation of yoghurts indicated that the yoghurt with CL-In
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received higher scores for overall acceptability than yoghurt containing lower DP
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inulin, whereas the different types of inulin did not significantly affect odour and
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colour of set-style yoghurt. Therefore, exploited as an effective prebiotic ingredient,
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the cross-linked inulin prepared could have significant application potential for
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improving textural characteristics and prolonging shelf-life of set-style yoghurt.
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Acknowledgements
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This work was financially supported by the special project for people's livelihood of Chongqing Municipal Science and Technology Commission
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[cstc2015shmszx0367] and the National Natural Science Foundation of China
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[31471581].
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References
Achanta, K., Aryana, K. J., & Boeneke, C. A. (2007). Fat free plain set yoghurts
M AN U
423
SC
422
424
fortified with various minerals. LWT - Food Science and Technology, 3, 424–
425
429.
426
Akalin, A. S., Fenderya, S., & Akbulut, N. (2004). Viability and activity of bifidobacteria in yoghurt containing fructooligosaccharide during refrigerated
428
storage. International Journal of Food Science and Technology, 39, 613–621.
429
TE D
427
Akalin, A. S., Gönç, S., Unal, G., & Fenderya, S. (2007). Effects of fructooligosaccharide and whey protein concentrate on the viability of starter
431
culture in reduced-fat probiotic yoghurt during storage. Journal of Food
433 434
AC C
432
EP
430
Science, 72, 222–227.
Akalin, A. S., Unal, G., Dinkci, N., & Hayaloglu, A. A. (2012). Microstructural, textural, and sensory characteristics of probiotic yoghurts fortified with
435
sodium calcium caseinate or whey protein concentrate. Journal of Dairy
436
Science, 95, 3617–3628. 21
ACCEPTED MANUSCRIPT 437
Akbari, M., Eskandari, M. H., Niakosari, M., & Bedeltavana, A. (2016). The effect of
438
inulin on the physicochemical properties and sensory attributes of low-fat ice
439
cream. International Dairy Journal, 57, 52–55.
441
AOAC. (2002). Official methods of analysis of the AOAC (15th edn.). Arlington, VI, USA: Association of Official Analytical Chemists.
RI PT
440
Aryana, K. J. & Mcgrew, P. (2007). Quality attributes of yoghurt with Lactobacillus
443
casei and various prebiotics. LWT - Food Science and Technology, 40, 1808–
444
1814.
M AN U
445
SC
442
Aryana, K. J., Plauche, S., Rao, R. M., Mcgrew, P., & Shah, N. P. (2007). Fat-free
446
plain yoghurt manufactured with inulins of various chain lengths and
447
Lactobacillus acidophilus. Journal of Food Science, 72, 79–84. Balthazar, C. F., Conte-Júnior, C. A., Moraes, J., Costa, M. P., Raices, R. S. L., Franco,
449
R. M., et al. (2016). Physicochemical evaluation of sheep milk yoghurts
450
containing different levels of inulin. Journal of Dairy Science, 99, 4160–4168.
453
EP
452
Balthazar, C. F., Gaze, L. V., Leandro, A. D. S. H., Pereira, C. S., Franco, R. M., Conte-Júnior, C. A., et al. (2015). Sensory evaluation of ovine milk yoghurt
AC C
451
TE D
448
with inulin addition. International Journal of Dairy Technology, 68, 281–290.
454
Balthazar, C. F., Silva, H. L. A., Cavalcanti, R. N., Esmerino, E. A., Cappato, L. P.,
455
Abud, Y. K. D., et al. (2017). Prebiotics addition in sheep milk ice cream: A
456
rheological, microstructural and sensory study. Journal of Functional Foods,
457
35, 564–573. 22
ACCEPTED MANUSCRIPT 458
Balthazar, C. F., Silva, H. L. A., Esmerino, E. A., Rocha, R. S., Moraes, J., Carmo, M.
459
A. V., et al. (2018). The addition of inulin and Lactobacillus casei 01 in sheep
460
milk ice cream. Food Chemistry, 246, 464–472. Canbulat, Z., & Ozcan, T. (2015). Effects of short-chain and long-chain inulin on the
RI PT
461 462
quality of probiotic yoghurt containing Lactobacillus rhamnosus. Journal of
463
Food Processing and Preservation, 39, 1251–1260.
Chiavaro, E., Vittadini, E., & Corradini, C. (2007). Physicochemical characterization
SC
464
and stability of inulin gels. European Food Research and Technology, 225,
466
85–94.
467
M AN U
465
Cruz, A. G., Antunes, A. E. C., Sousa, A. O. P., Faria, J. A. F., & Saad, S. M. I. (2009). Ice-cream as a probiotic food carrier. Food Research International, 42, 1233–
469
1239.
470
TE D
468
Glibowski, P., & Kowalska, A. (2012). Rheological, texture and sensory properties of kefir with high performance and native inulin. Journal of Food Engineering,
472
111, 299–304.
474 475 476
Glibowski, P., & Rybak, P. (2016). Rheological and sensory properties of stirred
AC C
473
EP
471
yoghurt with inulin-type fructans. International Journal of Dairy Technology, 69, 122–128.
Glibowski, P., & Zielińska, E. (2015). Physicochemical and sensory properties of
477
kefir containing inulin and oligofructose. International Journal of Dairy
478
Technology, 68, 602–607. 23
ACCEPTED MANUSCRIPT Guimarães, J. T., Silva, E. K., Alvarenga, V. O., Costa, A., Cunha, R. L., Freitas, M.
480
Q., et al. (2018a). Physicochemical changes and microbial inactivation after
481
high-intensity ultrasound processing of prebiotic whey beverage applying
482
different ultrasonic power levels. Ultrasonics – Sonochemistry, 44, 251–260.
483
RI PT
479
Guimarães, J. T., Silva, E. K., Costa, A. L. R., Cunha, R. L., Freitas, M. Q., Meireles, M. A. A., et al. (2018b). Manufacturing a prebiotic whey beverage exploring
485
the influence of degree of inulin polymerization. Food Hydrocolloids, 77,
486
787–795.
M AN U
487
SC
484
Guven, M., Yasar, K., Karaca, O. B., & Hayaloglu, A. A. (2005). The effect of inulin
488
as a fat replacer on the quality of set-type low-fat yoghurt manufacture.
489
International Journal of Dairy Technology, 58, 180–184. ISO. (2003). ISO 7889/IDF 117: Yoghurt - Enumeration of characteristic
TE D
490 491
microorganisms - Colony-count technique at 37 °C. Geneva, Switzerland
492
International Standardisation Organisation.
EP
494
Kip, P., Meyer, D., & Jellema, R. H. (2006). Inulins improve sensoric and textural properties of low-fat yoghurts. International Dairy Journal, 16, 1098–1103.
AC C
493
495
Kusuma, G. D., Paseephol, T., & Sherkat, F. (2009). Prebiotic and rheological effects
496
of Jerusalem artichoke inulin in low-fat yoghurt. Australian Journal of Dairy
497
Technology, 64, 159–163.
498
Lankaputhra, W. E., Shah, N. P., & Britz, M. L. (1996). Survival of bifidobacteria
499
during refrigerated storage in the presence of acid and hydrogen peroxide. 24
ACCEPTED MANUSCRIPT 500 501
Milchwissenschaft, 51, 65–69. Li, S. P., Hu, T., Chen, Y. L., Zheng, C. Y., Liu, T., Ma, G. H., et al. (2010). Cross-linked inulin as a potential plasma expander: Biochemical properties
503
and physiological characterization in a rabbit model. Carbohydrate Polymers,
504
82, 1054–1060.
RI PT
502
Li, Y., Ma, X., & Liu, X. (2018). Physicochemical and rheological properties of
506
cross-linked inulin with different degree of polymerization. Food
507
Hydrocolloids, in press.
M AN U
SC
505
508
Luo, D., Li, Y., Xu, B., Ren, G, Li, P., Li, X., et al. (2017). Effects of inulin with
509
different degree of polymerization on gelatinization and retrogradation of
510
wheat starch. Food Chemistry, 229, 35–45.
512
Meyer, D., Bayarri, S., Tárrega, A., & Costell, E. (2011). Inulin as texture modifier in
TE D
511
dairy products. Food Hydrocolloids, 25, 1881–1890. Perrin, S., Fougnies, C., Grill, J. P., Jacobs, H., & Schneider, F. (2002). Fermentation
514
of chicory fructo-oligosaccharides in mixtures of different degrees of
515
polymerization by three strains of bifidobacteria. Canadian Journal of
517 518
AC C
516
EP
513
Microbiology, 48, 759–763.
Ren, L., Jiang, M., Wang, L., Zhou, J., & Tong, J. (2012). A method for improving dispersion of starch nanocrystals in water through crosslinking modification
519
with sodium hexametaphosphate. Carbohydrate Polymers, 87, 1874–1876.
520
Rodríguez-García, J., Salvador, A., & Hernando, I. (2014). Replacing fat and sugar 25
ACCEPTED MANUSCRIPT 521
with inulin in cakes: bubble size distribution, physical and sensory properties.
522
Food and Bioprocess Technology, 7, 964–974.
524
Schreiner G. E. (1950). Determination of inulin by means of resorcinol. Proceedings of the Society for Experimental Biology and Medicine, 74, 117–120.
RI PT
523
Srisuvor, N., Chinprahast, N., Prakitchaiwattana, C., & Subhimaros, S. (2013). Effects
526
of inulin and polydextrose on physicochemical and sensory properties of
527
low-fat set yoghurt with probiotic-cultured banana purée. LWT - Food Science
528
and Technology, 51, 30–36. Tarrega, A., & Costell, E. (2005).
M AN U
529
SC
525
Effect of inulin addition on rheological and
530
sensory properties of fat-free starch-based dairy desserts. International Dairy
531
Journal, 16, 1104–1112.
Tiwari, A., Sharma, H. K., Kumar, N., & Kaur, M. (2015). The effect of inulin as a fat
TE D
532 533
replacer on the quality of low- fat ice cream. International Journal of Dairy
534
Technology, 68, 374–380.
537 538 539
EP
536
Villegas, B., & Costell, E. (2007). Flow behaviour of inulin–milk beverages. Influence of inulin average chain length and of milk fat content. International
AC C
535
Dairy Journal 17, 776–781.
Yi, H., Zhang, L., Hua, C., Sun, K., & Zhang, L. (2010). Extraction and enzymatic hydrolysis of inulin from Jerusalem artichoke and their effects on textural and
540
sensorial characteristics of yoghurt. Food and Bioprocess Technology, 3, 315–
541
319. 26
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Fig. 1. The molecular structural formula of cross-linked inulin (from: Li, Ma, & Liu, 2018).
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Table 1
2
Effect of polymerisation degree of inulin on acidity (g 100 g-1) of set-style yoghurt. a
3
Day 4
Day 7
Day 11
Control 0.86±0.01dC
0.86±0.01bC 0.87±0.02cC
Day 14
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Sample Day 1
0.90±0.01cB 0.93±0.02A
0.89±0.01bcB 0.87±0.03bB 0.89±0.01cB
0.98±0.01bA 0.99±0.02bA
CS-In
0.94±0.01aD
0.94±0.01aD 0.96±0.01aC
1.07±0.01aB 1.10±0.01aA
L-In
0.88±0.01cC
0.92±0.01aB 0.93±0.01bB
0.97±0.01bA 0.93±0.01cB
CL-In
0.90±0.01bC
0.94±0.01aB 0.95±0.01abB 0.98±0.01bA 0.97±0.01bA
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S-In
4
a
5
CS-In, yoghurts made with CS-In; L-In, yoghurts made with L-In; CL-In, yoghurts
6
made with CL-In. Mean values followed by different superscript lowercase and
7
uppercase letters in columns for different inulin types and in rows for time (day),
8
respectively, are significantly different (p ≤ 0.05).
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Treatments: Control, yoghurts made without inulin; S-In, yoghurts made with S-In;
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Table 2
11
Effect of polymerisation degree of inulin on syneresis (g 100 g-1) of set-style yoghurt.
12
a
Sample Day 1
Day 4
Day 7
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Day 11
Day 14
Control 19.31±0.90aC 19.73±0.81aC 21.08±0.84aC 23.16±1.16aB 25.50±1.25aA 15.93±0.51bC 17.38±0.76bB 17.89±0.49bB 17.57±0.70bB 19.20±0.88bA
CS-In
14.01±0.55cD 14.43±0.48cD 16.29±0.53cC 17.91±0.65bB 18.98±0.60bA
L-In
13.67±0.42dD 14.79±0.56cC 14.87±0.47dC 17.28±0.57bB 18.46±0.75bA
CL-In
12.54±0.81dB 13.86±0.72cA 14.57±0.53dA 15.02±0.82cA 15.06±0.45cA
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S-In
14
a
15
CS-In, yoghurts made with CS-In; L-In, yoghurts made with L-In; CL-In, yoghurts
16
made with CL-In. Mean values followed by different superscript lowercase and
17
uppercase letters in columns for different inulin types and in rows for time (day),
18
respectively, are significantly different (p ≤ 0.05).
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Treatments: Control, yoghurts made without inulin; S-In, yoghurts made with S-In;
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Table 3
22
Effect of polymerisation degree of inulin texture characteristics of set-style yoghurt. a
23
Cohesiveness ratio 1 0.41±0.03aB 7 0.42±0.07aA 14 0.35±0.04aA
0.40±0.05aAB 0.42±0.02aA 0.38±0.02aA
Adhesiveness (mJ) 1 3.67±0.25bC 7 4.63±0.06aC 14 4.80±0.20aC
4.33±0.15bB 4.63±0.15aC 4.87±0.12aBC
0.38±0.04aAB 0.41±0.02aA 0.37±0.04aA
L-In
4.52±0.17bB 4.94±0.24abBC 5.17±0.26aBC
CL-In
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99.67±3.51bB 104.52±4.26bB 103.67±3.51abB 107.33±3.92abAB 113.67±8.50aB 115.57±5.58aB
24
CS-In
108.00±5.00aB 121.67±5.51aA 113.67±9.50aB 130.33±10.50aA 118.33±4.5aB 133.67±5.51aA 0.38±0.02aAB 0.41±0.02aA 0.38±0.03aA
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Time Control (days) Firmness (g) 1 106.00±6.00bB 7 102.33±0.58bB 14 123.67±4.51aAB
4.43±0.15bB 5.60±0.20aB 5.63±0.25aB
0.34±0.02aA 0.38±0.02aA 0.35±0.03aA 6.63±0.35aA 7.03±0.75aA 7.27±0.80aA
25
a
26
CS-In, yoghurts made with CS-In; L-In, yoghurts made with L-In; CL-In, yoghurts
27
made with CL-In. Mean values followed by different superscript lowercase and
28
uppercase letters in columns for different inulin types and in rows for time (day),
29
respectively, are significantly different (p ≤ 0.05).
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Treatments: Control, yoghurts made without inulin; S-In, yoghurts made with S-In;
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Table 4
31
Bacterial counts (log cfu g-1) in various yoghurts with different types of inulin over a
32
storage period of 14 days. a L-In
CL-In
7.64±0.19bA 8.72±0.15aA 9.11±0.30aA 8.94±0.17aB 8.85±0.25aA
7.60±0.21bA 8.67±0.28aA 8.90±0.37aA 8.95±0.28aB 8.75±0.31aA
7.46±0.38cA 8.84±0.26bA 9.33±0.37abA 9.62±0.36aA 9.08±0.18bA
Lactobacillus delbrueckii ssp. bulgaricus 1 7.81±0.31bA 7.76±0.15bA 7.76±0.18bA 4 8.67±0.25aA 8.81±0.42aA 8.72±0.34aA 7 8.87±0.17aA 8.89±0.25aA 9.11±0.27aA 11 8.66±0.26aB 8.86±0.19aB 8.94±0.31aB 14 8.59±0.11aA 8.79±0.24aA 8.84±0.34aA
7.64±0.24bA 8.67±0.21aA 8.80±0.26aA 8.95±0.18aB 8.75±0.22aA
7.58±0.19cA 8.74±0.28bA 9.33±0.33abA 9.62±0.50aA 9.08±0.32abA
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Sampling Control S-In (day) Streptococcus thermophilus 1 7.82±0.28bA 7.76±0.28bA 4 8.69±0.23aA 8.81±0.19aA 7 8.77±0.15aA 8.88±0.25aA 11 8.74±0.13aB 8.86±0.12aB 14 8.66±0.32aA 8.79±0.55aA
33
a
34
CS-In, yoghurts made with CS-In; L-In, yoghurts made with L-In; CL-In, yoghurts
35
made with CL-In. Mean values followed by different superscript lowercase and
36
uppercase letters in columns for different inulin types and in rows for time (day),
37
respectively, are significantly different (p ≤ 0.05).
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Table 5
39
Colour characteristics and sensory evaluation of set-style yoghurts with different inulin preparations. a
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L*
a*
b*
Colour
Odour
Taste
Control S-In CS-In L-In CL-In
90.25±6.70a 95.82±0.35b 97.22±0.12b 95.56±0.26b 95.12±0.14b
–1.69±0.27a –1.51±0.06a –1.50±0.06a –1.53±0.03a –1.17±0.02b
7.17±1.64a 8.85±0.17b 9.19±0.25b 9.05±0.31b 11.13±0.08c
8.64±1.20a 8.77±1.47a 7.78±0.83a 8.37±1.03a 8.52±1.08a
8.06±1.14a 8.07±1.64a 7.69±1.01a 8.69±1.13a 8.56±1.35a
7.47±1.29a 7.69±1.38a 7.02±1.19a 7.64±1.49a 7.73±1.36a
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Texture and consistency 5.15±1.28a 7.43±1.35b 5.69±1.30ab 8.11±1.49b 7.72±1.60ab
Overall acceptability 6.98±0.72a 7.85±0.52ab 7.01±0.81ab 8.34±0.89ab 8.94±0.75b
40
a
41
with L-In; CL-In, yoghurts made with CL-In. Mean values followed by different superscript lowercase and uppercase letters in columns for
42
different inulin types and in rows for time (day), respectively, are significantly different (p ≤ 0.05).
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Treatments: Control, yoghurts made without inulin; S-In, yoghurts made with S-In; CS-In, yoghurts made with CS-In; L-In, yoghurts made
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S0958-6946(19)30052-4
DOI:
https://doi.org/10.1016/j.idairyj.2019.02.009
Reference:
INDA 4464
To appear in:
International Dairy Journal
Received Date: 31 October 2018 Revised Date:
17 February 2019
Accepted Date: 19 February 2019
Please cite this article as: Li, Y., Shabani, K.I., Qin, X., Yang, R., Jin, X., Ma, X., Liu, X., Effects of crosslinked inulin with different polymerisation degrees on physicochemical and sensory properties of setstyle yoghurt, International Dairy Journal, https://doi.org/10.1016/j.idairyj.2019.02.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of cross-linked inulin with different polymerisation degrees on
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physicochemical and sensory properties of set-style yoghurt
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Yao Li, Kinyoro Ibrahim Shabani, Xiaoli Qin, Rong Yang, Xuedong Jin, Xiaohan Ma,
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Xiong Liu*
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College of Food Science, Southwest University, 400715 Chongqing, China
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*Corresponding author. Tel.: +86 23 68250375
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E-mail address: [email protected] (X. Liu)
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ABSTRACT
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This work investigated effects of cross-linked inulin with different degrees of
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polymerisation (DP, average = 7 and 15) on physicochemical and sensory properties
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of set-style yoghurt. Compared with set-style yoghurt made with native inulin
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(average DP = 4 and 11), yoghurts with cross-linked inulin had higher acidity and
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lower syneresis values, with a shelf-life of 14 days. Supplementation of cross-linked
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inulin with higher DP resulted in enhanced firmness and adhesiveness of yoghurts. In
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addition, bacterial counts showed that yoghurts with cross-linked inulin exhibited
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longer retention of Streptococcus thermophilus and Lactobacillus delbrueckii ssp.
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bulgaricus cell viability than that with native inulin. Sensory evaluation indicated that
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yoghurt with cross-linked long-chain inulin received higher scores for overall
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acceptability than other samples. However, different types of inulin did not
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significantly affect odour and colour of set-style yoghurt. Consequently, cross-linked
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inulin prepared can be exploited as a prebiotic to prolong shelf-life of yoghurt.
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1.
Introduction
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Inulin is a polysaccharide composed of numerous fructose units and a terminal glucose unit. It can be exacted from various plants, such as onions, garlics, bananas
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and chicory roots. Native inulin has varying degrees of polymerisation (DP) ranging
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from 2 to 60 monosaccharide units, which depends on the source, harvest time and
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production process (Glibowski & Zielińska, 2015). Inulin preparations with different
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DP have varying physicochemical properties. Generally, long-chain inulin can act as a
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fat replacer or texture modifier due to its poor solubility and good viscosity stability,
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while short-chain inulin can contribute to improved mouthfeel due to good solubility
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and mild sweetness (Glibowski & Rybak, 2016). As a non-digestible dietary fibre and
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attractive prebiotic, inulin exhibits many beneficial physiological functions, such as
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antioxidant activity, ability to improve colonic microbiota, promoting mineral
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absorption, and regulating blood glucose and lipids (Balthazar et al., 2016).
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Based on nutritional and physicochemical properties, inulin has been widely
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used in an increasing number of food applications. Inulin has proven to be a
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promising fat substitute and to improve the functional potential in sheep milk ice
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cream formulation (Balthazar et al., 2017, 2018). Inulin can also be added for
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stabilisation in the formulation of whey beverage (Guimaraes et al., 2018a) and may
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enhance the viability of Lactobacillus in probiotic yoghurt during storage (Canbulat &
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Ozcan, 2015).
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However, native inulin (a mixture of short-chain and long-chain inulin) has usually been employed without classification in most recent studies, and the lack of
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certainty of the DP of inulin leads to variable effects on food products since inulin
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with different DP has different effects. Luo et al. (2017) reported that inulin with
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lower DP displayed stronger suppression of the retrogradation of wheat starch
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compared with higher DP inulin. Guimaraes et al. (2018b) showed that inulin with
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higher DP had better efficiency for beverage stabilisation. Nevertheless, there is still a
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lack of information about the effect of inulin with different DP on properties of other
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food products. Moreover, short-chain inulin may induce rapid renal excretion due to
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its low molecular weight. Cross-linking of inulin to increase its molecular weight is a
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strategy to avoid this disadvantage (Li et al. 2010). However, there is still no reported
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research exploring the influence of cross-linked inulin with different polymerisation
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degrees on quality attributes of food products, especially on such a popular dairy food
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as set-style yoghurt.
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Therefore, the present work was designed to prepare various set-style yoghurts containing native inulin or cross-linked inulin of different DP and aimed to evaluate
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the different effects of native inulin and cross-linked inulin on physiochemical
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properties and sensory properties of yoghurt, such as water retention, acidity,
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adhesiveness, firmness, colour, taste, and bacterial counts.
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2.
Materials and methods 4
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2.1.
Materials
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Native inulins (average DP < 10) were supplied by Xi’an Ruiling Technology Co., Ltd. (Xi’an, China). Four types of inulin with different DP, i.e., native inulin S-In
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(average DP = 4), native inulin L-In (average DP = 11), cross-linked inulin CS-In
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(average DP = 7), and cross-linked inulin CL-In (average DP = 15), were prepared in
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our laboratory by the method described in Section 2.2. Commercial yoghurt starter
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culture (YFL-702, Chr. Hansen, Denmark) containing Streptococcus thermophilus and
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Lactobacillus delbrueckii ssp. bulgaricus was used. Milk powder was obtained from
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Nestle Co., Ltd. (Switzerland). Peptone water was purchased from Qingdao Jisskang
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biochemical Co., Ltd. (China). Anhydrous ethanol, sodium hexametaphosphate (SH),
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hydrochloric acid, phenolphthalein, sodium hydroxide, glutaraldehyde, potassium
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dihydrogen phosphate, tertiary butyl alcohol were purchased from Kelong Chemical
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Reagent Factory (Chengdu, China). All reagents were of analytical grade.
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2.2.
Preparation of cross-linked inulin
Native inulin (16 g) was added to 80 mL distilled water at room temperature
and gently stirred until dissolved. Exactly 20 mL of anhydrous ethanol was added into the solution. The mixture solution was stored at 4 °C for 24 h, after which sediment 5
ACCEPTED MANUSCRIPT and liquid supernatant were separated by centrifugation at 8000 × g, 10 min. The
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sediment was lyophilised with vacuum freeze-drying equipment (Sihuan LGJ-25C,
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Beijing, China) at 5 Pa and –50 °C for 12 h; the product obtained was long-chain
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inulin (L-In) in native inulin. The liquid supernatant was condensed by rotary
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evaporation (Rongsheng RE-52CS, Shanghai, China) followed by lyophilising for 24
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h (5 Pa, –50 °C); the product obtained was short-chain inulin (S-In). Cross-linked
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inulin was prepared by a method similar to that of starch modification (Ren, Jiang,
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Wang, Zhou, & Tong, 2012). Typically, L-In (10 g) was dispersed in water (200 mL)
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with constant stirring for 1 h at 25 °C and then 1.1 g of cross-linker SH was added
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followed by the addition of Na2CO3 (2 M) to attain pH 10. Then the
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pre-polymerisation solution was polymerised with continuous stirring at 45 °C for 3.5
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h. Upon completion of the reaction, the pH of the suspension was adjusted to 7 with 2
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M
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centrifugation and washing with anhydrous ethanol. Finally, after lyophilising,
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cross-linked long-chain inulin (CL-In) was obtained. Cross-linked short-chain inulin
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(CS-In) was prepared in the same manner but with the S-In in place of L-In. The
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molecular structural formula of cross-linked inulin is illustrated in Fig. 1 (Li, Ma &
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Liu, 2018).
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HCl. The cross-linked inulin was then precipitated with 95% ethanol, followed by
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2.3.
Preparation of yoghurt
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Non-supplemented (inulin-free) control yoghurt was prepared by dispersing 16.0% (w/w) milk powder and 8% sugar in deionised water. Inulin-supplemented
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yoghurts were made from 16.0% (w/w) milk powder, 6% sugar and 2% corresponding
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inulin (respectively, S-In, L-In, CS-In, and CL-In) and deionised water. All
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ingredients were weighed in a flask and mixed with continuous agitation (Sile
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HD2004W, Shanghai, China) at 60 °C until completely dissolved. The reconstituted
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milk mixtures were homogenised (20 MPa, 60 °C) in a homogeniser (Nuoni
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GJJ-0.06/40, Zhejiang, China) and then flasks with samples were heated in a water
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bath at 95 °C for 15 min and then rapidly cooled in chilled water to 45 °C. After that,
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all the mixes were each inoculated with 1 g L-1 of yoghurt culture YFL-702, followed
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by incubation at 42 °C for 5 h. When acidity reached 0.8% lactic acid, the yoghurt
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was cooled to 4 °C.
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2.4.
Physicochemical determinations
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Acidity, expressed as g of lactic acid per 100 g of yoghurt, was determined on
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days 1, 4, 7, 11 and 14 according to the titration method of the Association of Official
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Analytical Chemists (AOAC, 2002).
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To measure syneresis (g per 100 g), a batch of prepared set-style yoghurt (25 g)
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was weighed in a centrifuge tube and then stored at 4 °C for 1, 4, 7, 11, and 14 days.
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Each sample was centrifuged at 3000 × g (Centrifuge 5810, Eppendorf, Germany) at 7
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fine mesh screen placed on top of a funnel. The clear supernatant was poured off, then
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weighed and expressed as percentage weight relative to the original weight of yoghurt
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(Srisuvor, Chinprahast, Prakitchaiwattana, & Subhimaros, 2013). All analyses were
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carried out in triplicate.
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Textural characteristics such as hardness, cohesiveness, and adhesiveness were evaluated using a texture analyser (CT-3; Brookfield, USA) equipped with a
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TA4/1000 cylindrical probe (38.1 mm diameter). The yoghurt samples prepared were
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stored at 4 °C for 24 h before analysis. The texture analysis circumstances were as
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follows: penetration depth, 30 mm; force, 5 g; probe speed during penetration, 1 mm
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s-1 (Tiwari, Sharma, Kumar, & Kaur, 2015). The samples, kept in original containers
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with a diameter of 70 mm, were penetrated at ambient temperature (10 ± 1 °C). Two
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cycles were applied; the values for texture attributes were obtained from the resulting
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curve using the Texture Pro CT equipment software. The following parameters were
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quantified: firmness as defined as the maximal peak value recorded in the first
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immersion into the sample; cohesiveness defined as the ratio of positive force area
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during the second immersion cycle to that of the first immersion cycle; and
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adhesiveness, or the negative area for the first cycle, representing the work necessary
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to pull the probe away from the sample.
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The colours of set-style yoghurts stored at 4 °C for 24 h were measured with a
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colorimeter (Minolta, Spectrophotometer CM-3500d, Japan). The colorimeter used L* 8
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(lightness), a* (redness) and b* (yellowness) scales, and operating conditions were
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illuminant D65 and 2° sample-observer. Colour was measured in triplicate for each
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sample (Akbari, Eskandari, Niakosari, & Bedeltavana, 2016).
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2.5.
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Determination of bacterial counts
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A representative sample of yoghurt (1 g) was suspended in 9 mL of sterile 0.1% (w/v) peptone water (Jisskang, Qingdao, China) and subsequently serially diluted to
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10-7. Counts of S. thermophilus (ST) were enumerated using the pour plate technique
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after aerobic incubation at 37 ºC for 72 h on M17 agar (Merck, USA) and
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enumeration of L. delbrueckii ssp. bulgaricus (LB) was performed using MRS agar
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(Merck, USA) incubated at 37 °C for 72 h under anaerobic conditions, according to
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the technique for yoghurt enumeration of characteristic microorganism colony count
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described by International Organisation for Standardisation (ISO, 2003).
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2.6.
Sensory evaluation
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Sensory evaluations were carried out 3 days after production and by a group of
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20 panellists (10 females and 10 males, aged between 17 and 38 years old) who were
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trained in sensory analysis of dairy products. Panellists evaluated the samples and
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recorded their perceptions by making marks on a ten-score grades as described by 9
ACCEPTED MANUSCRIPT Glibowski and Kowalska (2012). The properties evaluated included: colour (1 –
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unnatural colour, 10 – no criticism), taste (1 – bad, 10 – no criticism), odour (0 –
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undetectable, 10 – very intensive), texture and consistency (0 – liquid, 10 – very
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thick), and overall acceptability (0 – dislike extremely, 10 – like extremely).
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Definitions for the descriptors describing the colour, taste, odour, texture and
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consistency of set-style yoghurt were developed by panel consensus using reference
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samples. Sample defects and descriptors such as acid, sweet, bitter, acetaldehyde taste,
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milky, creamy, grainy, off-flavour, ropy and gritty texture, whey syneresis, and
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lumpiness were recorded. Each sample (30 mL) was scored individually, and samples
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were presented to the panellists in random and balanced order in white plastic cups
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coded with three-digit random numbers. Mineral water was provided for mouth
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rinsing in between samples.
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2.7.
Statistical analysis
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All experiments were conducted in triplicate for each sample, and results were
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expressed as mean ± standard deviation. All data were statistically compared between
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groups using one-way analysis of variance. Significant differences among the mean
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values were determined by Duncan’s multiple range test with p < 0.05.
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3.
Results and discussion 10
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3.1.
Analysis of acidity
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The titratable acidity values of the stored set-style yoghurt are presented in
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Table 1. An increase in titratable acidity was observed over storage time, due to more
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lactic acid production and the higher acidity in the yoghurt caused by continuous
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fermentation and stimulated growth of lactic bacteria, which often results in greater
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contraction of the casein micelle matrix and increased expulsion of whey (Achanta,
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Aryana, & Boeneke, 2007). Thus, the shelf-life of set-style yoghurt is often limited by
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excessive acidification during storage (Akalin, Gönç, Unal, & Fenderya, 2007).
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Compared with yoghurt containing inulin, the control yoghurt had the lowest acidity value, which agrees well with that reported by Aryana et al. (2007) and Guven
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et al. (2005). According to research of Perrin et al. (2002), the acidity of yoghurt
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containing lower DP inulin was higher than that of others, due to the faster
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consumption by the probiotic bacteria which could increase lactate production.
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However, data in Table 1 are not consistent with this trend, as these results showed
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that set-style yoghurt containing 2% cross-linked inulin (CS-In or CL-In) had
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significantly higher acidity than that with corresponding native inulin (S-In or L-In),
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while the CS-In yoghurt had a higher acidity than the CL-In yoghurt. Consequently,
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acidity values of yoghurts were mainly influenced by the introduction of acid
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phosphate groups and DP of inulin.
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According to the result of a study of acid stability of inulin (Li et al., 2018), breakdown and hydrolysis would occur for the cross-linked inulin in acidic
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environment (pH <6) and inulin with higher DP was more difficult to transform into
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reducing sugar. The pH value of yoghurt is about 4, so cross-linked inulin should
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partly break down to reducing sugar and phosphoric acid in this acidic condition, and
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the generated phosphoric acid would further increase the yoghurt acidity.
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3.2.
Analysis of syneresis
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Syneresis value could reflect the water-holding capability of yoghurt product during storage. The syneresis values of yoghurt samples over storage are presented in
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Table 2. Both inulin polymerisation degree and storage time had significant effects on
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syneresis. There was a steady increase in syneresis with storage time for all yoghurt
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samples, including the control, which may be explained in terms of the increase in
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acidity during storage causing the casein micelle matrix to contract and extrude more
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whey (Canbulat & Ozcan, 2015). The extent of syneresis was affected by the addition
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of inulin. Yoghurt samples made with inulin underwent less syneresis than the control,
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which may be explained by the ability of inulin molecules to bind water, preventing
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free movement of water, and to complex with protein aggregates and provide stability
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to the protein network. In fact, many studies have shown decreased syneresis of
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yoghurt following the addition of inulin (Meyer, Bayarri, Tárrega, & Costell, 2011).
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Interestingly, yoghurts made with CL-In had significantly lower syneresis values than those of yoghurt made with other inulins, which suggests that CL-In
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possibly had a better water-holding capacity than L-In, S-In, and S-In, due to the
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higher DP. This result was consistent with that of Aryana and Mcgrew (2007) who
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reported that long-chain prebiotic had a better water-holding capacity than that of
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short-chain prebiotic in yoghurt. A good-quality yoghurt should hold water without
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syneresis. Therefore, the utility of CL-In in this study to act as a food stabiliser could
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contribute to the yoghurt gel structure, preventing fracture and whey-off.
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3.3.
Analysis of textural characteristics
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Table 3 shows the effect of adding different types of inulin at a level of 2% on
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textural parameters of set-style yoghurts. Some significant differences in textural
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parameters were observed among different yoghurt samples. The addition of S-In and
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CS-In, composed of short-chain inulin, decreased the firmness of control yoghurt
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compared to L-In and CL-In with higher DP. Set yoghurt made with cross-linked
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inulin (CS-In and CL-In) had higher firmness than that with corresponding native
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inulin (S-In and L-In), which can be explained by the fact that the viscosity of inulin
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increases with the degree of polymerisation and cross-linked inulin had a higher
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viscosity and water-binding capacity than native inulin, which caused enhanced
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molecular flow resistance and decreased molecular mobility (Akalin, Unal, Dinkci, &
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firmness of yoghurt. Some studies have shown decreased firmness of yoghurt with
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added inulin (Kusuma, Paseephol, & Sherkat, 2009), and the lower firmness of
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inulin-supplemented yoghurts may be attributed to short-chain inulin not generating
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strong viscosity and possibly interfering with the formation of the protein network,
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resulting in softening of yoghurt. Therefore, the DP of added inulin had an important
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influence on firmness of yoghurt, a result consistent with that of Glibowski &
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Kowalska (2012).
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The same observations were made for adhesiveness, which is defined as the work required to overcoming the attraction forces between the surface of yoghurt and
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the probe surface (Chiavaro, Vittadini, & Corradini, 2007). Yoghurt containing inulin
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with higher DP had greater adhesiveness. The presence of CL-In significantly
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increased the work required to remove yoghurt that adheres to the probe. This result is
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in agreement with that of Villegas and Costell (2007), who showed that addition of
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inulin with higher DP resulted in significantly enhanced adhesiveness of whole milk.
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Values of firmness and adhesiveness of yoghurt made with CL-In were higher than
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those of the yoghurts made with other inulin types, which could be attributed to that
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inulin of higher DP generating a higher viscosity and water-binding capacity.
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Cohesiveness ratio, expressed as values between 0 and 1, show the ability for
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rebuilding the structure of the sample. The highest value of cohesiveness was shown
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by control yoghurt, with slightly lower cohesiveness by yoghurt with inulin. A lower 14
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cohesiveness ratio was observed in yoghurt with CL-In, which could be due to inulin
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of higher DP being dispersed within the casein micelles, interfering with protein
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matrix formation (Meyer et al., 2011).
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3.4.
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Counts of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus
Changes in counts of S. thermophilus and L. delbrueckii ssp. bulgaricus over a
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storage period of 14 days are presented in Table 4. Storage time markedly influenced
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the levels of S. thermophilus and L. delbrueckii ssp. bulgaricus in CL-In yoghurt
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throughout 14 days of storage, while statistical differences of counts for other sample
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groups (the control, CS, CS-In and CL) could be observed only between day 1 and
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day 4. This is in agreement with results of Balthazar et al. (2016), who reported
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significant changes occurred in S. thermophilus and L. delbrueckii ssp. bulgaricus
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counts during the storage of yoghurt. Although very few statistical differences were
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observed between samples (control and different inulin types) for each day of storage,
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overall, the counts of S. thermophilus and L. delbrueckii ssp. bulgaricus in yoghurts
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with inulin were slightly higher than that of the control after storage of day 4. The
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result was consistent with Akalin, Fenderya, and Akbulut (2004), who reported higher
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counts of two commercial strains of bifidobacteria in yoghurts containing
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fructo-oligosaccharide compared with yoghurts without this prebiotic. At the
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thermophilus and L. delbrueckii ssp. bulgaricus, varying from 7.46-7.82 log cfu g-1
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for S. thermophilus and 7.58-7.81 log cfu g-1 for L. delbrueckii ssp. bulgaricus. The
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numbers of S. thermophilus and L. delbrueckii ssp. bulgaricus in control, S-In, CS-In
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treatments remained marginally increased up to day 7, but slowly declined over the
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following week, whereas the counts of S. thermophilus and L. delbrueckii ssp.
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bulgaricus in L-In, CL-In yoghurts slightly rose up to day 11 and then decreased. By
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day 14, the populations of S. thermophilus and L. delbrueckii ssp. bulgaricus in all
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yoghurt samples increased compared with the beginning, while the increase in
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yoghurts containing CL-In was most noticeable (1.48 log cfu g-1 for S. thermophilus
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and 1.5 log cfu g-1 for L. delbrueckii ssp. bulgaricus).
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of Lactobacillus acidophilus in yoghurts supplemented with long-chain inulin than
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yoghurts with short-chain inulin at the end of storage. Yoghurts containing S-In and
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CS-In showed slightly higher counts in both S. thermophilus and L. delbrueckii ssp.
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bulgaricus than that of L-In and CL-In on day 1 and day 4, suggesting that
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supplementation with inulin with an increased DP led to a decreased rate of
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consumption of inulin by S. thermophilus and L. delbrueckii ssp. bulgaricus.
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According to Akalin et al. (2007), the prebiotic activity of inulin was dependent on
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DP of inulin and shorter-chain inulin is more commonly used as a prebiotic. However,
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this trend was reversed at subsequent storage up to day 11 and day 14, as the S.
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CL-In were higher than that of S-In, CS-In and the control. This may be explained by
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the high acidity of yoghurts with short-chain inulin, consistent with the findings of
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Lankaputhra, Shah, and Britz (1996) and Cruz et al. (2009) that high yoghurt acidity
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negatively affected the viability of probiotic bacteria. Overall, yoghurts with added
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CL-In exhibited longer retention of S. thermophilus and L. delbrueckii ssp. bulgaricus
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cell viability than yoghurts made with other DP inulin.
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3.5.
Colour characteristics
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The colour values (L*, a*, b*) of different yoghurts are summarised in Table 5. For different yoghurts, a* values varied between –1.69 and –1.17, and b* values varied
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from 7.17 to 11.13 while, overall, both a* and b* values increased with higher inulin
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DP. Addition of inulin gave slightly higher L*, b* and a* values than the control. These
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changes in colour may be associated with inulin molecules interacting with casein
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micelles, which along with the fat globules, are responsible for the diffusion of
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incident light. In contrast, Aryana et al. (2007) reported that addition of 1.5% (w/w)
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inulin in yoghurts did not have any significant effect on L∗, a∗, or b∗ values. The
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different result can be explained by the higher concentration of casein and fat globules
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in this matrix and higher inulin amount used in this work. However, Balthazar et al.
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(2015) observed slightly higher L*, b* and a* values for ovine milk yoghurt with inulin,
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ACCEPTED MANUSCRIPT in agreement with this work. In addition, acid hydrolysis of inulin can probably occur
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in yoghurt containing inulin due to the presence of lactic acid formed by the starter
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culture and adequate fermentation temperature (42 ± 1 °C), interfering with the colour
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of yoghurts. Schreiner (1950) reported a colorimetric method for the determination of
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inulin by means of resorcinol, due to the ability of inulin to change the medium colour
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after acid hydrolysis above 40 °C. Although all yoghurt samples appeared white to the
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naked eye, the instrument picked up slight green and yellow colours.
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3.6.
Sensory evaluation
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Sensory evaluation to complement the physicochemical properties of the yoghurt samples were performed (Table 5). No significant effect of inulin on odour,
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colour and taste was observed. However, significant differences were observed
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between the sensory evaluations regarding the texture and consistency and overall
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acceptability of yoghurt samples. For overall acceptability, the sensory scores of the
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yoghurt containing CL-In and L-In were slightly higher than those of the control
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yoghurt and the yoghurt containing S-In and CS-In. In addition, the panellists deemed
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that S-In and CS-In yoghurts were slightly sweeter than CL-In and CL-In yoghurts,
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which tasted more smooth and creamy, this indicating that taste and texture of yoghurt,
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to a certain extent, was influenced by the presence of inulin. This is in agreement with
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the result of Canbulat and Ozcan (2015), who reported that short-chain inulin has 30%
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in chain length between short-chain inulin and long-chain inulin account for their
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distinctly different functionality attributes, short-chain inulin possesses functional
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qualities similar to sugar or glucose syrup, etc., while long-chain inulin has been used
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successfully to replace fat in dairy products and baked goods.
379
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Several authors have studied the replacement of sugar and fat with different
types of inulin in food products. Tarrega and Costell (2005) showed that adding inulin
381
to fat-free dairy model desserts significantly increased sweetness compared with those
382
with no addition. Rodríguez-García, Salvador, and Hernando (2014) reported the good
383
functionality of short-chain inulin as replacers for sugar in low-sugar cakes and
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long-chain inulin as replacers for fat in low-fat cakes. Another interesting observation
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was that of worse texture and consistency scores given to CS-In in comparison with
386
other types of inulin yoghurts. Similar results have been reported in some studies
387
concerning yoghurts with inulin. Aryana et al. (2007) reported that inulin chain length
388
had insignificant effect on colour and appearance of fat-free yoghurt, but that
389
medium-chain and long-chain inulin gave yoghurt better sensory properties. Yi,
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Zhang, Hua, Sun, and Zhang (2010) found that inulin and its hydrolysate could
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partially replace sucrose in yoghurt. Kip, Meyer, and Jellema (2006) observed that
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moderately long-chain inulin could be used successfully to improve the creamy
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mouthfeel of low-fat yoghurt, whereas Guven et al. (2005) found that excessive
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addition of inulin in fat-free yoghurt negatively influenced some physical properties
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of yoghurt, i.e., whey separation, consistency and organoleptic scores.
396 397
4.
Conclusions
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Compared with set-style yoghurt made with native inulin (S-In and L-In), the
yoghurt samples supplemented with cross-linked inulin (CS-In and CL-In) had higher
401
acidity and lower syneresis values during the observed shelf-life of 14 days,
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indicating that inulin with higher DP had better water-holding capacity.
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Supplementation of cross-linked inulin with higher DP resulted in enhanced firmness and adhesiveness of yoghurt. In addition, bacterial counts indicated that the
405
addition of cross-linked inulin as a prebiotic to the yoghurt led to longer retention of S.
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thermophilus and L. delbrueckii ssp. bulgaricus cell viability during storage.
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Moreover, sensory evaluation of yoghurts indicated that the yoghurt with CL-In
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received higher scores for overall acceptability than yoghurt containing lower DP
409
inulin, whereas the different types of inulin did not significantly affect odour and
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colour of set-style yoghurt. Therefore, exploited as an effective prebiotic ingredient,
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the cross-linked inulin prepared could have significant application potential for
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improving textural characteristics and prolonging shelf-life of set-style yoghurt.
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Acknowledgements
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This work was financially supported by the special project for people's livelihood of Chongqing Municipal Science and Technology Commission
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[cstc2015shmszx0367] and the National Natural Science Foundation of China
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[31471581].
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References
Achanta, K., Aryana, K. J., & Boeneke, C. A. (2007). Fat free plain set yoghurts
M AN U
423
SC
422
424
fortified with various minerals. LWT - Food Science and Technology, 3, 424–
425
429.
426
Akalin, A. S., Fenderya, S., & Akbulut, N. (2004). Viability and activity of bifidobacteria in yoghurt containing fructooligosaccharide during refrigerated
428
storage. International Journal of Food Science and Technology, 39, 613–621.
429
TE D
427
Akalin, A. S., Gönç, S., Unal, G., & Fenderya, S. (2007). Effects of fructooligosaccharide and whey protein concentrate on the viability of starter
431
culture in reduced-fat probiotic yoghurt during storage. Journal of Food
433 434
AC C
432
EP
430
Science, 72, 222–227.
Akalin, A. S., Unal, G., Dinkci, N., & Hayaloglu, A. A. (2012). Microstructural, textural, and sensory characteristics of probiotic yoghurts fortified with
435
sodium calcium caseinate or whey protein concentrate. Journal of Dairy
436
Science, 95, 3617–3628. 21
ACCEPTED MANUSCRIPT 437
Akbari, M., Eskandari, M. H., Niakosari, M., & Bedeltavana, A. (2016). The effect of
438
inulin on the physicochemical properties and sensory attributes of low-fat ice
439
cream. International Dairy Journal, 57, 52–55.
441
AOAC. (2002). Official methods of analysis of the AOAC (15th edn.). Arlington, VI, USA: Association of Official Analytical Chemists.
RI PT
440
Aryana, K. J. & Mcgrew, P. (2007). Quality attributes of yoghurt with Lactobacillus
443
casei and various prebiotics. LWT - Food Science and Technology, 40, 1808–
444
1814.
M AN U
445
SC
442
Aryana, K. J., Plauche, S., Rao, R. M., Mcgrew, P., & Shah, N. P. (2007). Fat-free
446
plain yoghurt manufactured with inulins of various chain lengths and
447
Lactobacillus acidophilus. Journal of Food Science, 72, 79–84. Balthazar, C. F., Conte-Júnior, C. A., Moraes, J., Costa, M. P., Raices, R. S. L., Franco,
449
R. M., et al. (2016). Physicochemical evaluation of sheep milk yoghurts
450
containing different levels of inulin. Journal of Dairy Science, 99, 4160–4168.
453
EP
452
Balthazar, C. F., Gaze, L. V., Leandro, A. D. S. H., Pereira, C. S., Franco, R. M., Conte-Júnior, C. A., et al. (2015). Sensory evaluation of ovine milk yoghurt
AC C
451
TE D
448
with inulin addition. International Journal of Dairy Technology, 68, 281–290.
454
Balthazar, C. F., Silva, H. L. A., Cavalcanti, R. N., Esmerino, E. A., Cappato, L. P.,
455
Abud, Y. K. D., et al. (2017). Prebiotics addition in sheep milk ice cream: A
456
rheological, microstructural and sensory study. Journal of Functional Foods,
457
35, 564–573. 22
ACCEPTED MANUSCRIPT 458
Balthazar, C. F., Silva, H. L. A., Esmerino, E. A., Rocha, R. S., Moraes, J., Carmo, M.
459
A. V., et al. (2018). The addition of inulin and Lactobacillus casei 01 in sheep
460
milk ice cream. Food Chemistry, 246, 464–472. Canbulat, Z., & Ozcan, T. (2015). Effects of short-chain and long-chain inulin on the
RI PT
461 462
quality of probiotic yoghurt containing Lactobacillus rhamnosus. Journal of
463
Food Processing and Preservation, 39, 1251–1260.
Chiavaro, E., Vittadini, E., & Corradini, C. (2007). Physicochemical characterization
SC
464
and stability of inulin gels. European Food Research and Technology, 225,
466
85–94.
467
M AN U
465
Cruz, A. G., Antunes, A. E. C., Sousa, A. O. P., Faria, J. A. F., & Saad, S. M. I. (2009). Ice-cream as a probiotic food carrier. Food Research International, 42, 1233–
469
1239.
470
TE D
468
Glibowski, P., & Kowalska, A. (2012). Rheological, texture and sensory properties of kefir with high performance and native inulin. Journal of Food Engineering,
472
111, 299–304.
474 475 476
Glibowski, P., & Rybak, P. (2016). Rheological and sensory properties of stirred
AC C
473
EP
471
yoghurt with inulin-type fructans. International Journal of Dairy Technology, 69, 122–128.
Glibowski, P., & Zielińska, E. (2015). Physicochemical and sensory properties of
477
kefir containing inulin and oligofructose. International Journal of Dairy
478
Technology, 68, 602–607. 23
ACCEPTED MANUSCRIPT Guimarães, J. T., Silva, E. K., Alvarenga, V. O., Costa, A., Cunha, R. L., Freitas, M.
480
Q., et al. (2018a). Physicochemical changes and microbial inactivation after
481
high-intensity ultrasound processing of prebiotic whey beverage applying
482
different ultrasonic power levels. Ultrasonics – Sonochemistry, 44, 251–260.
483
RI PT
479
Guimarães, J. T., Silva, E. K., Costa, A. L. R., Cunha, R. L., Freitas, M. Q., Meireles, M. A. A., et al. (2018b). Manufacturing a prebiotic whey beverage exploring
485
the influence of degree of inulin polymerization. Food Hydrocolloids, 77,
486
787–795.
M AN U
487
SC
484
Guven, M., Yasar, K., Karaca, O. B., & Hayaloglu, A. A. (2005). The effect of inulin
488
as a fat replacer on the quality of set-type low-fat yoghurt manufacture.
489
International Journal of Dairy Technology, 58, 180–184. ISO. (2003). ISO 7889/IDF 117: Yoghurt - Enumeration of characteristic
TE D
490 491
microorganisms - Colony-count technique at 37 °C. Geneva, Switzerland
492
International Standardisation Organisation.
EP
494
Kip, P., Meyer, D., & Jellema, R. H. (2006). Inulins improve sensoric and textural properties of low-fat yoghurts. International Dairy Journal, 16, 1098–1103.
AC C
493
495
Kusuma, G. D., Paseephol, T., & Sherkat, F. (2009). Prebiotic and rheological effects
496
of Jerusalem artichoke inulin in low-fat yoghurt. Australian Journal of Dairy
497
Technology, 64, 159–163.
498
Lankaputhra, W. E., Shah, N. P., & Britz, M. L. (1996). Survival of bifidobacteria
499
during refrigerated storage in the presence of acid and hydrogen peroxide. 24
ACCEPTED MANUSCRIPT 500 501
Milchwissenschaft, 51, 65–69. Li, S. P., Hu, T., Chen, Y. L., Zheng, C. Y., Liu, T., Ma, G. H., et al. (2010). Cross-linked inulin as a potential plasma expander: Biochemical properties
503
and physiological characterization in a rabbit model. Carbohydrate Polymers,
504
82, 1054–1060.
RI PT
502
Li, Y., Ma, X., & Liu, X. (2018). Physicochemical and rheological properties of
506
cross-linked inulin with different degree of polymerization. Food
507
Hydrocolloids, in press.
M AN U
SC
505
508
Luo, D., Li, Y., Xu, B., Ren, G, Li, P., Li, X., et al. (2017). Effects of inulin with
509
different degree of polymerization on gelatinization and retrogradation of
510
wheat starch. Food Chemistry, 229, 35–45.
512
Meyer, D., Bayarri, S., Tárrega, A., & Costell, E. (2011). Inulin as texture modifier in
TE D
511
dairy products. Food Hydrocolloids, 25, 1881–1890. Perrin, S., Fougnies, C., Grill, J. P., Jacobs, H., & Schneider, F. (2002). Fermentation
514
of chicory fructo-oligosaccharides in mixtures of different degrees of
515
polymerization by three strains of bifidobacteria. Canadian Journal of
517 518
AC C
516
EP
513
Microbiology, 48, 759–763.
Ren, L., Jiang, M., Wang, L., Zhou, J., & Tong, J. (2012). A method for improving dispersion of starch nanocrystals in water through crosslinking modification
519
with sodium hexametaphosphate. Carbohydrate Polymers, 87, 1874–1876.
520
Rodríguez-García, J., Salvador, A., & Hernando, I. (2014). Replacing fat and sugar 25
ACCEPTED MANUSCRIPT 521
with inulin in cakes: bubble size distribution, physical and sensory properties.
522
Food and Bioprocess Technology, 7, 964–974.
524
Schreiner G. E. (1950). Determination of inulin by means of resorcinol. Proceedings of the Society for Experimental Biology and Medicine, 74, 117–120.
RI PT
523
Srisuvor, N., Chinprahast, N., Prakitchaiwattana, C., & Subhimaros, S. (2013). Effects
526
of inulin and polydextrose on physicochemical and sensory properties of
527
low-fat set yoghurt with probiotic-cultured banana purée. LWT - Food Science
528
and Technology, 51, 30–36. Tarrega, A., & Costell, E. (2005).
M AN U
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SC
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Effect of inulin addition on rheological and
530
sensory properties of fat-free starch-based dairy desserts. International Dairy
531
Journal, 16, 1104–1112.
Tiwari, A., Sharma, H. K., Kumar, N., & Kaur, M. (2015). The effect of inulin as a fat
TE D
532 533
replacer on the quality of low- fat ice cream. International Journal of Dairy
534
Technology, 68, 374–380.
537 538 539
EP
536
Villegas, B., & Costell, E. (2007). Flow behaviour of inulin–milk beverages. Influence of inulin average chain length and of milk fat content. International
AC C
535
Dairy Journal 17, 776–781.
Yi, H., Zhang, L., Hua, C., Sun, K., & Zhang, L. (2010). Extraction and enzymatic hydrolysis of inulin from Jerusalem artichoke and their effects on textural and
540
sensorial characteristics of yoghurt. Food and Bioprocess Technology, 3, 315–
541
319. 26
ACCEPTED MANUSCRIPT Figure legend
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Fig. 1. The molecular structural formula of cross-linked inulin (from: Li, Ma, & Liu, 2018).
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Table 1
2
Effect of polymerisation degree of inulin on acidity (g 100 g-1) of set-style yoghurt. a
3
Day 4
Day 7
Day 11
Control 0.86±0.01dC
0.86±0.01bC 0.87±0.02cC
Day 14
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Sample Day 1
0.90±0.01cB 0.93±0.02A
0.89±0.01bcB 0.87±0.03bB 0.89±0.01cB
0.98±0.01bA 0.99±0.02bA
CS-In
0.94±0.01aD
0.94±0.01aD 0.96±0.01aC
1.07±0.01aB 1.10±0.01aA
L-In
0.88±0.01cC
0.92±0.01aB 0.93±0.01bB
0.97±0.01bA 0.93±0.01cB
CL-In
0.90±0.01bC
0.94±0.01aB 0.95±0.01abB 0.98±0.01bA 0.97±0.01bA
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S-In
4
a
5
CS-In, yoghurts made with CS-In; L-In, yoghurts made with L-In; CL-In, yoghurts
6
made with CL-In. Mean values followed by different superscript lowercase and
7
uppercase letters in columns for different inulin types and in rows for time (day),
8
respectively, are significantly different (p ≤ 0.05).
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Treatments: Control, yoghurts made without inulin; S-In, yoghurts made with S-In;
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Table 2
11
Effect of polymerisation degree of inulin on syneresis (g 100 g-1) of set-style yoghurt.
12
a
Sample Day 1
Day 4
Day 7
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Day 11
Day 14
Control 19.31±0.90aC 19.73±0.81aC 21.08±0.84aC 23.16±1.16aB 25.50±1.25aA 15.93±0.51bC 17.38±0.76bB 17.89±0.49bB 17.57±0.70bB 19.20±0.88bA
CS-In
14.01±0.55cD 14.43±0.48cD 16.29±0.53cC 17.91±0.65bB 18.98±0.60bA
L-In
13.67±0.42dD 14.79±0.56cC 14.87±0.47dC 17.28±0.57bB 18.46±0.75bA
CL-In
12.54±0.81dB 13.86±0.72cA 14.57±0.53dA 15.02±0.82cA 15.06±0.45cA
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S-In
14
a
15
CS-In, yoghurts made with CS-In; L-In, yoghurts made with L-In; CL-In, yoghurts
16
made with CL-In. Mean values followed by different superscript lowercase and
17
uppercase letters in columns for different inulin types and in rows for time (day),
18
respectively, are significantly different (p ≤ 0.05).
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Treatments: Control, yoghurts made without inulin; S-In, yoghurts made with S-In;
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Table 3
22
Effect of polymerisation degree of inulin texture characteristics of set-style yoghurt. a
23
Cohesiveness ratio 1 0.41±0.03aB 7 0.42±0.07aA 14 0.35±0.04aA
0.40±0.05aAB 0.42±0.02aA 0.38±0.02aA
Adhesiveness (mJ) 1 3.67±0.25bC 7 4.63±0.06aC 14 4.80±0.20aC
4.33±0.15bB 4.63±0.15aC 4.87±0.12aBC
0.38±0.04aAB 0.41±0.02aA 0.37±0.04aA
L-In
4.52±0.17bB 4.94±0.24abBC 5.17±0.26aBC
CL-In
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99.67±3.51bB 104.52±4.26bB 103.67±3.51abB 107.33±3.92abAB 113.67±8.50aB 115.57±5.58aB
24
CS-In
108.00±5.00aB 121.67±5.51aA 113.67±9.50aB 130.33±10.50aA 118.33±4.5aB 133.67±5.51aA 0.38±0.02aAB 0.41±0.02aA 0.38±0.03aA
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Time Control (days) Firmness (g) 1 106.00±6.00bB 7 102.33±0.58bB 14 123.67±4.51aAB
4.43±0.15bB 5.60±0.20aB 5.63±0.25aB
0.34±0.02aA 0.38±0.02aA 0.35±0.03aA 6.63±0.35aA 7.03±0.75aA 7.27±0.80aA
25
a
26
CS-In, yoghurts made with CS-In; L-In, yoghurts made with L-In; CL-In, yoghurts
27
made with CL-In. Mean values followed by different superscript lowercase and
28
uppercase letters in columns for different inulin types and in rows for time (day),
29
respectively, are significantly different (p ≤ 0.05).
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Treatments: Control, yoghurts made without inulin; S-In, yoghurts made with S-In;
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Table 4
31
Bacterial counts (log cfu g-1) in various yoghurts with different types of inulin over a
32
storage period of 14 days. a L-In
CL-In
7.64±0.19bA 8.72±0.15aA 9.11±0.30aA 8.94±0.17aB 8.85±0.25aA
7.60±0.21bA 8.67±0.28aA 8.90±0.37aA 8.95±0.28aB 8.75±0.31aA
7.46±0.38cA 8.84±0.26bA 9.33±0.37abA 9.62±0.36aA 9.08±0.18bA
Lactobacillus delbrueckii ssp. bulgaricus 1 7.81±0.31bA 7.76±0.15bA 7.76±0.18bA 4 8.67±0.25aA 8.81±0.42aA 8.72±0.34aA 7 8.87±0.17aA 8.89±0.25aA 9.11±0.27aA 11 8.66±0.26aB 8.86±0.19aB 8.94±0.31aB 14 8.59±0.11aA 8.79±0.24aA 8.84±0.34aA
7.64±0.24bA 8.67±0.21aA 8.80±0.26aA 8.95±0.18aB 8.75±0.22aA
7.58±0.19cA 8.74±0.28bA 9.33±0.33abA 9.62±0.50aA 9.08±0.32abA
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Sampling Control S-In (day) Streptococcus thermophilus 1 7.82±0.28bA 7.76±0.28bA 4 8.69±0.23aA 8.81±0.19aA 7 8.77±0.15aA 8.88±0.25aA 11 8.74±0.13aB 8.86±0.12aB 14 8.66±0.32aA 8.79±0.55aA
33
a
34
CS-In, yoghurts made with CS-In; L-In, yoghurts made with L-In; CL-In, yoghurts
35
made with CL-In. Mean values followed by different superscript lowercase and
36
uppercase letters in columns for different inulin types and in rows for time (day),
37
respectively, are significantly different (p ≤ 0.05).
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Table 5
39
Colour characteristics and sensory evaluation of set-style yoghurts with different inulin preparations. a
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L*
a*
b*
Colour
Odour
Taste
Control S-In CS-In L-In CL-In
90.25±6.70a 95.82±0.35b 97.22±0.12b 95.56±0.26b 95.12±0.14b
–1.69±0.27a –1.51±0.06a –1.50±0.06a –1.53±0.03a –1.17±0.02b
7.17±1.64a 8.85±0.17b 9.19±0.25b 9.05±0.31b 11.13±0.08c
8.64±1.20a 8.77±1.47a 7.78±0.83a 8.37±1.03a 8.52±1.08a
8.06±1.14a 8.07±1.64a 7.69±1.01a 8.69±1.13a 8.56±1.35a
7.47±1.29a 7.69±1.38a 7.02±1.19a 7.64±1.49a 7.73±1.36a
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Texture and consistency 5.15±1.28a 7.43±1.35b 5.69±1.30ab 8.11±1.49b 7.72±1.60ab
Overall acceptability 6.98±0.72a 7.85±0.52ab 7.01±0.81ab 8.34±0.89ab 8.94±0.75b
40
a
41
with L-In; CL-In, yoghurts made with CL-In. Mean values followed by different superscript lowercase and uppercase letters in columns for
42
different inulin types and in rows for time (day), respectively, are significantly different (p ≤ 0.05).
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Treatments: Control, yoghurts made without inulin; S-In, yoghurts made with S-In; CS-In, yoghurts made with CS-In; L-In, yoghurts made
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