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Blackwell Publishing, Ltd.


Effects of Lactobacillus rhamnosus GG addition in ice cream

Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche (DISTAM), Universit degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy

A 24 full factorial experimental design was applied to verify the effects of Lactobacillus rhamnosus GG (LGG) addition in retail-manufactured ice cream stored at two different freezing temperatures (16C and 28C) and containing two different levels of sugar (1522%) and fat (510%). In addition to microbial counts, the pH, acidity, viscosity of the mixes and functional properties of the ice creams were evaluated. Both fresh and frozen-thawed LGG cells underwent preliminary resistance tests to bile, antibiotics and acidity. The LGG strain proved to be highly resistant to most of the stress factors. When the micro-organism was added to ice cream mixes in a quantity of 108 cfu/g, it did not change the overrun, rmness or melting behaviour of the nished product. Regardless of formulation, no count decay of LGG cells was observed in ice cream stored for up to 1 year. Keywords Lactobacillus rhamnosus GG, Ice cream, Probiotics, Stress factor.

I N T RO D U C T I O N The production of dairy products with added lactobacilli and bidobacteria cultures is still expanding in the food market, as several studies have proved the benecial effects on the consumer deriving from the ingestion of bacteria named probiotics (Fuller 1992; Fuller 1994). According to a generally accepted denition, the term probiotic refers to live and safe micro-organisms in a food or a supplement that survive the passage through the gut, improve the microbial balance in the large bowel and exert a positive impact on the host. More specically, the metabolic activities of selected strains belonging to the Lactobacillus genus allow the body to enhance the digestion of lactose, decrease the cholesterol level in the blood serum, prevent intestinal disorders and stimulate the immune function (Havenaar et al. 1992; Gilliland and Walker 1989). Many authors (Lee and Salminen 1995; Saxelin 1997; Vinderola et al. 2000; Oliveira et al. 2001) suggest that an ingestion of 106109 viable cells per day is needed to develop benecial effects for humans, and so the survival rate of the probiotic bacteria in foods should be kept as high as possible prior to consumption. Ice cream seems suitable for delivering probiotics in the human diet. Nevertheless, freezing and thawing may seriously damage the cells, causing death (lethal effect), or inhibition of multiplication and/or interruption of metabolic activity (sublethal effect) (Speck and Cowman 1968; Davies and
*Author for correspondence. E-mail:

*Author for correspondence. E-mail: 2005 Society of Dairy Technology

Obafemi 1985), which could defeat the potential advantages of probiotics. Frozen yogurt and ice cream prepared using fermented mixes have already been made under controlled conditions with the purpose of ensuring that an adequate content of viable micro-organisms survive the freezing operation and storage (Kaul and Mathur 1982; Hekmat and McMahon 1992; Christiansen et al. 1996; Inoue et al. 1998; Hagen and Narvhus 1999). On the other hand, many studies have proved that the mortality of lactobacilli increases with storage time and preservation temperatures of close to zero (Gibson et al. 1965; Smittle et al. 1974; Thunell et al. 1984; Foschino et al. 1996). Lactobacillus rhamnosus GG (LGG) (Valio Ltd, Helsinki, Finland) is one of the extensively studied strains with well-documented probiotic properties: it is known to colonize the intestine and to be active against organisms causing travellers diarrhoea and rotavirus infection in children (Salminen et al. 1998; Ouwehand and Salminen 1998; Saxelin 2001). The aim of this investigation was to study the inuence on ice cream characteristics of LGG addition in nonfermented retail-manufactured ice cream, and to verify the effects of different levels of sugar and fat on strain survival during ice cream storage. Moreover, the resistance of fresh and frozen-thawed LGG cells to some chemical stress factors were studied. Results were compared with those obtained in a previous work on Lactobacillus johnsonii La1 (Alamprese et al. 2002).


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Bacterial strain and culture conditions LGG (ATCC 53103) was kindly supplied by Dr Maija Saxelin (Valio Ltd, Helsinki, Finland). Stock culture was kept at 30C in cryogenic vials containing MRS (de Man, Rogosa and Sharpe) broth (Difco, Becton Dickinson France S. A., Le Pont de Claix, France) and glycerol (20% v/v). For the experiments, the strain was cultured in MRS broth at 37C overnight. The sensitivity stress tests were performed both on the overnight culture and on the same culture after storage at 16C for 1 month and thawing at room temperature. Sensitivity stress tests Acidity resistance, bile sensitivity and susceptibility to antibiotics were investigated in accordance with Alamprese et al. (2002). Ingredients for ice cream production The pasteurized skim milk and pasteurized cream (35% fat) used in the ice cream mixes were commercial products (Centrale del Latte di Milano, Milano, Italy). The solid ingredients used in the experiment were: sucrose (Eridania, Italy), glucose (Nuova Tradizione S.r.l., Italy), Nespray skim milk powder (Nestl Food Services, Nestl Italiana S.p.A., Milano, Italy); fatty acid mono- and diglycerides (E471), sodium alginate (E401) and locust bean gum (E410), all by Nuova Tradizione S.r.l., used as emulsiers and stabilizers. Preparation of culture A fermenter containing 2 l MRS broth was inoculated with 0.2% overnight LGG culture. After incubation at 37C for 16 h, cells were collected by centrifugation (4500 g for 30 min at 4C), washed twice with Ringer solution (Merck), suspended in about 1 l pasteurized ice cream mix and treated with UltraTurrax T25 (Janke & Kunkel, Germany) for 15 min at 13 500 r.p.m. in order to break cellular chains. This treatment is necessary in order to avoid subsequent overestimation of the number of cfu/g, which may be a result of the breaking up of cellular chains which can occur during freezingthawing of the product (Thunell et al. 1984; Foschino et al. 1992). Ice cream production A Pastomaster 60 Tronic (Carpigiani S.r.l., Anzola Emilia, Italy) was used to prepare the ice cream mix and pasteurize the batch. The probiotic culture was added after cooling to 4C, in order to achieve approximately 108 cfu/g. After ageing for 24 h at 4C, the mix was frozen in a Labotronic 2030 batch freezer (Carpigiani S.r.l.). Ice cream formulations and technological specications are reported by Alamprese et al. (2002).
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A full factorial design 24 was implemented. Four different mixes were formulated, each with a different fat (5% and 10%) and added sugar content (15% and 22%). For each mix, two versions of the ice cream were produced, with and without LGG cells, which were stored at two different temperatures (16C and 28C), in order to simulate both retailmanufactured and industrial conditions. Samples were identied with codes, in which the rst number is the percentage of added sugars, the second number is the percentage of fat, and the letter a or p indicates the absence or the presence of micro-organisms.

Mixes and ice cream analyses Ice cream mixes were analysed for pH, acidity and viscosity. Viscosity was determined at 4C using a Bohlin VOR Rheometer (Bohlin Reologi, Lund, Sweden). The analysis was carried out with a C25 measuring system and a torsion bar of 20.83 g cm. Results (Pas) are expressed as apparent viscosity determined at a shear rate of 292/s, as the average of three replicates. Some quality indexes (rmness, melting behaviour and overrun) of ice creams were evaluated, together with acidity and probiotics counts (Alamprese et al. 2002). Sensorial analysis A triangle method complying with the UNI U590A2520 standard (1999) was applied to compare ice cream with LGG cells and an ice cream control made without probiotics. In each test run, 30 judges evaluated the product. The analysis was carried out on an intermediate ice cream formulation, containing 7.5% fat and 18.5% added sugars. Statistical analysis The analysis of effects was carried out using unscrambler 6.11 (Camo ASA, Norway). systat 5.03 for Windows (Systat Inc., USA) was used to perform one-way anova.

Acid and bile sensitivity When selecting a probiotic culture to be used as a dietary adjunct, acid and bile resistance have to be considered (Bolin et al. 1997) since microbial cells have to overcome the stomach barrier and survive in the intestinal environment. Table 1 reports the results of the acid resistance test for the fresh and the frozen-thawed LGG cultures. The behaviour of the LGG strain in an acid environment is very similar to that observed for L. johnsonii La1 (Alamprese et al. 2002): after just 1 h of incubation at pH 1.5, the microbial count decreases signicantly (P < 0.01) by 4 log cycles, either for the fresh and for the frozen-thawed LGG cultures. For pH 2.5

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Table 1 Results of the acidity resistance test on Lactobacillus rhamnosus GG Time (h) 0 1 2 3 0 1 2 3 0 1 2 3 0 1 2 3 LGG fresh count (cfu/ml) 2.8 108 < 104 < 104 < 104 3.5 108 2.9 108 3.8 108 4.6 108 3.1 108 4.3 108 3.1 108 3.7 108 3.2 108 3.9 108 3.5 108 5.3 108 LGG frozen-thawed count (cfu/ml) < 104 < 104 < 104 < 104 3.5 108 1.5 108 4.7 107 6.3 107 2.2 108 1.5 108 1.4 108 1.6 108 1.5 108 1.8 108 1.2 108 1.4 108

Table 3 Results of the agar overlay diffusion test Amount (g) 2 5 10 20 50 2 5 10 20 50 5 15 30 50 80 2 5 10 20 50 5 15 30 50 80 1 3 5 10 20 50 150 300 500 800 Inhibition zone diameter (mm) 12 17 19 S 21 24 9 11 14 MS 16 20 21 24 S 26 30 R R 8 10 R 11 14 R

Antibiotic Penicillin G

pH 1.5







and 3.5, the number of cfu/ml is not signicantly different from the control (pH 6.5) after up to 3 h of incubation. For the same strain, Prasad et al. (1998) reported a survival rate of about 30% at pH 3.0 after 3 h. Results of LGG strain counts after plating both fresh and frozen-thawed cells with different percentages of bile are shown in Table 2. Unlike ndings for the La1 strain, no concentration level of bile caused a signicant reduction in the initial fresh and frozen-thawed cell counts, with the exception of the fresh culture plated at a 2.0% concentration of bile, for which the survival rate was signicantly lower (P < 0.05). The survival rate of fresh LGG strain to bile found by Prasad et al. (1998) was 80% for 0.4% bile concentration and 40% for 0.8% and 1.0% bile concentrations.




S = susceptible; MS = moderately susceptible; R = resistant

Table 2 Results of the bile sensitivity test on Lactobacillus rhamnosus GG Bile (%w/v) 0 0.1 0.2 0.4 0.8 1.2 1.6 2.0
a, b

LGG fresh count (cfu/ml) 2.8 108 a 2.6 108 a 3.4 108 a 3.1 108 a 2.9 108 a 2.9 108 a 1.6 108 a 5.0 107 b

LGG frozen-thawed count (cfu/ml) 3.0 108 a 3.1 108 a 3.4 108 a 3.7 108 a 2.8 108 a 3.3 108 a 2.7 108 a 2.3 108 a

= Means in the same column without a common subscript are signicantly different (P < 0.05)

Susceptibility to antibiotics Table 3 reports the results of the antibiotics resistance test, carried out by the agar overlay diffusion method on fresh cells. In addition to the antibiotics concentrations investigated by Charteris et al. (1998), other concentrations were tested. The LGG strain proved to be susceptible to penicillin G and cephalothin; moderately susceptible to ampicillin; and resistant to bacitracin, vancomycin, cexime and polymyxin. The results reported by Saxelin (2001) conrm the susceptibility to ampicillin and the vancomycin resistance, indicating a minimum inhibitory concentration (MIC) of 0.50 g/ml and > 258 g/ml, respectively. Actually, as reported by Tynkkynen et al. (1998), the species L. rhamnosus is intrinsically resistant to vancomycin due to the special structure of its cell wall peptidoglycan precursors.


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Table 4 Results (mean and SD) of physico-chemical analyses on aged mix samples Acidity (meq/100 g) SD 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 Mean 1.4 1.7 1.5 1.9 1.6 1.9 1.4 1.7 SD 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

pH Aged mix 15/5 a 15/5 p 15/10 a 15/10 p 22/5 a 22/5 p 22/10 a 22/10 p Mean 6.60 6.41 6.62 6.40 6.59 6.42 6.58 6.37

Viscosity (mPas) Mean 33.5 35.3 45.4 43.5 46.1 45.0 63.2 59.4 SD 0.3 0.8 1.0 0.1 1.4 0.9 2.0 1.8

Note: Mix code denotes sugar content/fat content then presence (p) or absence (a) of the culture

Assessment of mix and ice cream characteristics The most common quality indexes of mixes and ice creams were evaluated. Tables 4 and 5 report the results of the analyses carried out on aged mixes and on ice cream samples stored at 16C and 28C. Results of the analysis of effects of the four variables studied in the experiment (micro-organisms, sugar, fat and storage temperature) and the interactions between them are reported in Table 6. The presence of LGG cells decreases pH and viscosity in aged mix samples, whereas, obviously, mix viscosity increases when sugar and fat levels

Table 5 Results (mean and SD) of physical analyses performed on ice creams stored for 5 days at 16C and 28C and samples acidity analysed up to 90 days of storage Overrun Storage (%) temperature Mean SD (C) 16 16 16 16 16 16 16 16 28 28 28 28 28 28 28 28 29.0 27.0 23.7 24.5 24.9 26.2 27.2 24.0 25.5 27.5 22.6 23.9 26.7 24.9 20.7 27.3 1.1 5.2 2.9 1.4 0.3 2.0 0.8 1.0 1.8 4.3 1.0 1.9 3.3 1.3 0.6 0.9 Firmness (kg) Mean 2.63 3.76 2.69 2.52 0.69 0.53 0.38 0.41 5.64 4.46 3.87 3.09 0.75 0.75 0.50 0.51 SD 0.79 0.80 0.99 0.57 0.17 0.14 0.14 0.08 0.83 0.71 0.10 0.54 0.13 0.11 0.08 0.10 Melting rate (g/min) Mean 2.3 2.1 1.0 0.7 2.4 2.5 1.8 2.1 2.4 2.3 0.8 0.7 2.5 2.8 1.9 2.1 SD 0.3 0.3 0.1 0.1 0.1 0.1 0.3 0.5 0.1 0.1 0.1 0.2 0.3 0.1 0.2 0.4 Melting start (min) Acidity (meq/100 g) 0 days 30 days 90 days

Sample 15/5 a 15/5 p 15/10 a 15/10 p 22/5 a 22/5 p 22/10 a 22/10 p 15/5 a 15/5 p 15/10 a 15/10 p 22/5 a 22/5 p 22/10 a 22/10 p

Mean SD Mean SD Mean SD Mean SD 21.9 23.5 23.6 26.1 20.8 18.9 15.2 15.3 23.4 22.5 19.7 20.7 21.8 20.0 15.7 12.9 1.1 1.4 1.6 1.0 0.6 4.2 1.0 2.3 1.1 2.2 3.3 6.2 0.1 1.0 0.6 2.4 1.5 1.8 1.5 1.9 1.6 1.9 1.4 1.8 1.5 1.8 1.5 1.9 1.6 1.9 1.4 1.8 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 1.5 1.9 1.5 1.9 1.6 1.9 1.6 1.8 1.5 1.9 1.5 1.9 1.5 1.9 1.6 1.8 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 1.6 2.0 1.5 2.0 1.6 2.0 1.4 1.8 1.6 2.0 1.6 1.9 1.5 2.0 1.4 1.7 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1

Table 6 Analysis of the effectsa of four variables on ice cream production: micro-organisms, sugar, fat and storage temperature Mix Variable Microbes (A) Sugars (B) Fat (C) Temperature (D) AB AC AD BC BD CD

Ice cream Acidity NS NS NS NS NS NS Viscosity +++ +++ +++ Overrun NS NS NS NS NS NS NS NS NS NS Firmness NS ns ns ns ns ns ns Melting rate NS +++ NS +++ NS NS +++ NS ++ Melting start NS NS NS NS +

pH ++

Signicance level: not signicant, NS; P < 0.05, +/; P < 0.01, ++/ ; P < 0.001, +++/

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are higher. The functional properties of ice cream are not changed by the addition of LGG strain. Sugar and fat contents affect rmness and melting behaviour, but not the overrun of the ice creams. Data conrmed the results obtained in the work done with the La1 strain (Alamprese et al. 2002), except for ice cream overrun, which in the previous paper appeared to be correlated to fat content. This study has veried that ice cream with a higher fat content is softer and has a slower melting rate, as already observed by Roland et al. (1999). Some interactions have also been found between pairs of variables, and these particularly inuenced the mix characteristics and the melting behaviour.

LGG strain survival rate in retailmanufactured ice cream Table 7 shows the results of counts and the survival rate of LGG strain in mixes and newly produced ice creams. Mix 15/5 p contains a signicantly (P < 0.05) lower number of micro-organisms than mixes 15/10 p and 22/5 p, although the gure remains high. After freezing (ice cream 0 day), counts decreased signicantly in samples 15/10 p (P < 0.05) and 22/5 p (P < 0.05) by around 0.2 0.3 logarithmic units. This reduction is similar to that obtained in the previous work (Alamprese et al. 2002), but lower than that reported by other

authors (Hekmat and McMahon 1992; Christiansen et al. 1996; Hagen and Narvhus 1999). Table 8 shows the survival rate of LGG in ice cream stored at 16C for 30 days and 28C for up to 1 year. Samples 15/5 p and 22/5 p stored at 16C did not show signicant differences at any point during the whole storage period, whereas signicant variations of survival rate were found for samples 15/10 p and 22/10 p. After 30 days of storage at 16C, there was no signicant difference between the various samples of ice cream. After 3, 7 and 14 days, the variations, although signicant, cannot be ascribed to the different formulations. After 365 days at 28C, regardless of the formulation, LGG strain counts do not change signicantly as regards to the ice cream newly produced, although uctuations can be observed during the storage time. In this case too, where signicant differences were observed in the samples after the same storage time, they did not seem to be correlated to fat and sugar concentration. The considerable variations observed in the survival rates cannot be attributed to inhomogeneous cell distribution in the ice cream mix, since this is shaken for 10 s every 30 min during ageing. Probably the Ultra Turrax treatment carried out on the inoculum culture does not totally separate all

Table 7 Counts of Lactobacillus rhamnosus GG in mixes and newly produced ice creams and survival rate Sample Mix (cfu/g) Ice cream 0 days (cfu/g) Survival rate (%)

15/5 p 1.3 10 1.1 108 a A 85


15/10 p 2.4 10 1.6 108 ab B 48


22/5 p 2.3 10 1.1 108 ab B 67


22/10 p 2.1 108 ab A 2.1 108 b A 100


Means on the same line without a common subscript are signicantly different (P < 0.05) Means in the same column without a common subscript are signicantly different (P < 0.05)

Table 8 Survival rate (%) of Lactobacillus rhamnosus GG in ice cream samples after storage at 16C and 28C vs newly produced ice cream Storage temperature (C) 16 16 16 16 28 28 28 28 Storage days 0 100a 100ab 100a 100b 100a 100ab 100a 100bc 3 157a B 73a A 99a AB 84b A 70a A 90a AB 155a B 81abc AB 7 130a AB 98ab AB 161a B 83b A 133a A 94a A 133a A 70abc A 14 106a B 89ab AB 136a B 51a A 98a AB 113ab AB 149a B 54a A 30 116a A 148b A 138a A 73ab A 129a A 115ab A 99a A 89abc A 60 120 240 365

Sample 15/5 p 15/10 p 22/5 p 22/10 p 15/5 p 15/10 p 22/5 p 22/10 p


109a A 85a A 103a A 114c A

101a A 75a A 102a A 80abc A

122a A 173b A 112a A 108bc A

148a B 88a A 155a B 62ab A

Means on the same line without a common subscript are signicantly different (P < 0.05) Means in the same column and at the same temperature storage without a common subscript are signicantly different (P < 0.05)


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the cellular clumps, which are instead broken up during storage at low temperatures and thawing operation (Thunell et al. 1984; Foschino et al. 1992).

Sensorial analysis The sensorial analysis was carried out on an ice cream formulation with intermediate sugar and fat content, in order to average the inuence of the two components. The triangle method highlighted a signicant (P < 0.01) difference between the ice cream with LGG cells and a control ice cream produced without micro-organisms. Most ice cream is, however, avoured and this could probably hide the probiotic off avor.
CONCLUSIONS As already shown in previous work carried out with L. johnsonii La1 (Alamprese et al. 2002), this study has conrmed that it is possible to produce unfermented retail-manufactured ice cream containing probiotic bacteria, since the microorganism survival rate is high for up to 1 year of storage at 28C, regardless of the formulation. Also, preservation at 16C, as usually occurs for retail-manufactured product in ice cream shops, does not impair the survival of micro-organisms. Moreover, the addition of the LGG strain does not alter the structural characteristics of the product. Furthermore, results show that both fresh and frozenthawed LGG cells proved to be highly resistant to most of the chemical stress factors tested. The manufacture of probiotic ice creams may be an interesting choice for infants and adults who dislike fermented milks and cheeses. For individuals intolerant to some components of milk (e.g. lactose, proteins), probiotic vegetable ice cream might be suggested. REFERENCES
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