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Volume 7, Issue 5 2011 Article 8
International Journal of Food
Engineering
Effects of Lyophilization and Use of
Probiotics on Donkey’s Milk Nutritional
Characteristics
Silvia Vincenzetti, University of Camerino
Michele Savini, Synbiotec
Cinzia Cecchini, University of Camerino
Daniela Micozzi, University of Camerino
Francesco Carpi, University of Camerino
Alberto Vita, University of Camerino
Paolo Polidori, University of Camerino
Recommended Citation:
Vincenzetti, Silvia; Savini, Michele; Cecchini, Cinzia; Micozzi, Daniela; Carpi, Francesco; Vita,
Alberto; and Polidori, Paolo (2011) "Effects of Lyophilization and Use of Probiotics on
Donkey’s Milk Nutritional Characteristics," International Journal of Food Engineering: Vol. 7:
Iss. 5, Article 8.
DOI: 10.2202/1556-3758.2032
Available at: http://www.bepress.com/ijfe/vol7/iss5/art8
©2011 Berkeley Electronic Press. All rights reserved.
Effects of Lyophilization and Use of
Probiotics on Donkey’s Milk Nutritional
Characteristics
Silvia Vincenzetti, Michele Savini, Cinzia Cecchini, Daniela Micozzi, Francesco
Carpi, Alberto Vita, and Paolo Polidori
Abstract
Cow milk protein allergy (CMPA) is an abnormal IgE-mediated reaction to cow milk
proteins. Donkey’s milk could be considered suitable for feeding young children affected by
severe IgE-mediated CMPA because its nutritional properties and composition are very close to
human milk. Since donkey’s milk is available during a limited range of months during the year, it
may be useful to find better storage conditions for this product. This study investigated the effects
of the lyophilization treatment on donkey’s milk nutritional characteristics, and the results were
compared with those obtained on fresh and frozen milk. Nutritional properties of lyophilized
donkey’s milk remained basically unchanged compared with fresh milk. Two different probiotic
strains were added to lyophilized donkey’s milk, and their viability was evaluated after milk
reconstitution. The results obtained confirmed the possibility of producing a probiotic infant
formula with beneficial properties using donkey’s milk as raw material.
KEYWORDS: donkey’s milk, protein fractions, lyophilized milk, vitamin C, probiotic
Author Notes: This research was supported by a grant of the Italian Ministry of Agriculture,
principle investigator Dr. Paolo Polidori.
INTRODUCTION
Cow milk protein allergy (CMPA) is an abnormal immunological reaction to cow
milk proteins, resulting in immediate IgE-mediated reactions. Among infants, the
incidence of CMPA vary from 2.0% and 7.5% (Hill et al.1986). Different is the
cow milk protein intolerance that not involves the immune system. Furthermore
CMPA can develop also in infant that are partially or exclusively breast-fed, when
cow’s milk proteins are introduced into the feeding regimen. It is important an
early diagnosis and an adequate treatment that decrease the risk of impaired
growth of the child. Clinical manifestations of CMPA include gastrointestinal,
respiratory, cutaneous as well as systemic anaphylactic symptoms (Bahna et
al.1983). Cow’s milk contains more than 20 proteins that cause allergic reactions.
The main proteins in cow’s milk are caseins ((α-, β-, and κ-casein) and
whey proteins ((α-lactalbumin, β-lactoglobulin, bovine serum albumin, and
immunoglobulin), nevertheless the main allergens in cow’s milk are caseins
(mainly αs1- and β-caseins) followed by β-lactoglobulin and α-lactalbumin in
minor extent (Docena et al.1996; Bernard et al.1998; Järvinen et al. 2001).
Children that show clinical manifestations of CMPA may be fed with alternative
cow milk substitutes such as infant milk formula in which the proteins are
hydrolyzed by proteolytic enzymes to develop a number of casein, whey or soy
protein hydrolyzates (Terracciano et al. 2002). Extensively hydrolyzed formula
(EHF) often has a poor palatability but is used as first choice in children with
CMPA before using other formula (Terracciano et al. 2002). Amino acid-based
formula (AABF) is used in EHF allergic children (Kelly et al. 1995). Finally soy
formula are used offering equivalent nutritional benefits of EHF but are more
palatability; nevertheless it has been shown that 17–47% of milk allergic infants
can have adverse reactions to soy.
In order to find a better substitute to mother’s milk, when it is not possible
to breast feeding and when there is not the possibility to use a cow’s milk
alternative, several authors considered the clinical use of milk from different
animals: goat (Muraro et al. 2002; Restani et al. 2002), sheep (Dean et al. 1993;
Restani et al. 2002); mare and donkey (Carroccio et al. 2000; Iacono et al. 1992;
Monti et al. 2007). The cross-reactivity between milk proteins from different
mammalian species was object of several studies (Prieels et al. 1975; Carroccio et
al. 1999; Restani et al. 2002). In particular a strong cross-reactivity was shown
between sera of children with CMPA and milk proteins from other mammalian
species such as sheep, goat and buffalo (Restani et al. 1999) whereas a weak
cross-reactivity was observed with milk from mares and donkey. Donkey’s milk
could be considered suitable for feeding young children affected by severe IgE-
mediated cow milk allergy and it has been proposed as an alternative to cow’s
milk for children affected by cow’s milk protein intolerance when it is not
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Vincenzetti et al.: Donkey’s Milk Nutritional Characteristics
Published by Berkeley Electronic Press, 2011
possible breast feeding (Carroccio et al. 2000; Iacono et al. 1992; Monti et al.
2007).
The low allergenicity of donkey’s milk is mainly due to the low casein
content (Vincenzetti et al. 2007) that is characteristic of this milk and that is very
close to the casein content determined in human milk. About the caseins
composition in donkey’s milk, recent works (Vincenzetti et al. 2008; Criscione et
al. 2009; Bertino et al. 2010) showed the presence of of αS1- and β-caseins that
are present in different phosphorylated and glycosylated forms, while the presence
of κ-casein and αS2-casein are present in this milk but in very small amounts,
differently to dairy cow’s milk (Bertino et al. 2010; Creamer 2003).
Donkey’s milk whey proteins represent the 35-50% of the nitrogen
fraction whereas in bovine milk only the 20% (Herrouin et al. 2000). The total
whey protein content in donkey’s milk is 7.50 mg/ml (Vincenzetti et al. 2008)
very close to that found in the human milk (8.0 mg/ml) but higher with respect to
bovine milk (4.5 mg/ml), as stated by Martin and Grosclaude (1993). Among
whey proteins, α-lactalbumin concentration in donkey’s milk is 1.8 mg/ml
(Vincenzetti et al. 2008), very close to human milk (1.6 mg/ml); donkey α-
lactalbumin was identified as two isoforms both of them glycosylated (Bertino et
al. 2010). The high lysozyme content found in donkey’s milk (Vincenzetti et al.
2008) may be responsible of the low microbial load found in donkey’s milk
(Salimei et al. 2004) and could be useful to prevent intestine infections in infants.
Since donkey’s milk supply is limited in a range of a few months during
the year, because the fertility of donkey female is strictly connected with
photoperiod, it may be useful to find the better storage conditions for donkey’s
milk since in literature little is known about this topic. Until now in literature
there is only one work in which is reported the changes of microflora of donkey’s
milk during prolonged storage at 4°C and 20°C (Zhang et al. 2008).
The study on the consequences of a specific technological treatment on
milk, regarding the chemical-physical properties of protein fraction, brought to the
definition of quality indicators that may be related to modifications in the milk
protein structure induced by the treatment itself. The most important milk proteins
used to study the impact of thermal treatment in milk are whey proteins; in
particular some components such as β-lactoglobulin, α-lactalbumin and serum
albumin may be evaluated by chemical, chromatographic and electrophoretic
methods (Resmini et al. 1988; Pagliarini et al. 1990; Morales et al. 2000, Resmini
et al. 1989). Furthermore the assay of specific enzymes (such as lactoperoxidase)
that possess different thermal stability, may be used as indicators for controlling
the heat processing of milk (Barrett et al. 1999; Claeys et al. 2001).
In a previous work we evaluated the effects on the protein fractions of
donkey’s milk of a particular storage temperature (-20°C), and of a specific
technological process called “spray-drying” in which milk is subjected to a strong
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DOI: 10.2202/1556-3758.2032
thermal treatment necessary to produce powdered milk (Polidori and Vincenzetti,
2010). The object of this study was to investigate the effects of the lyophilization
(freeze-drying) treatment on donkey’s milk nutritional characteristics (protein
modifications and vitamin C content) and the results were compared with those
obtained from fresh and frozen milk. Furthermore the content of L-ascorbic acid,
commonly known as vitamin C, was determined for the first time in donkey’s
milk. This important vitamin is present in human colostrum and milk and has a lot
of biochemical functions such as maintenance of a natural barrier against
infection, stimulation of leukocytes for their phagocytic and anti-microbial
activity. A study conducted on human milk demonstrated that vitamin C may be
associated with a reduced risk of atopy in high-risk infants (Hoppu et al. 2005).
Vitamin C is also necessary for the synthesis of collagen, therefore it is critical to
the development of infants, it is also an effective antioxidant, because it can
protect indispensable molecules in the body from damage by free radicals and
reactive oxygen species generated during normal metabolism. For growth,
development and survival, infants need an optimum supply of vitamin C.
In some studies conducted in human milk in order to determine the
ascorbic acid content during the handling and storage of the milk (Hanna et al.
2004; Buss et al. 2001), it was reported that storage at both refrigerator and
freezer temperatures led to a significant decrease of the ascorbic acid levels. In the
present study the content of ascorbic acid in donkey’s milk was also evaluated
during storage of milk obtained by a lyophilization process and after a freezing
treatment.
Finally on the donkey’s milk before the lyophilization process were added
two different probiotic strains and their viability was evaluated after milk
reconstitution, in order to evaluate the possibility of using donkey’s milk for
probiotic purposes and consequently using this milk as a functional beverage also
in the diets of adults.
MATERIALS AND METHODS
Preparation of donkey’s milk samples
Bulk milk obtained from twenty Martina Franca breed pluriparous asses in mid-
stage of lactation was used. Skimmed milk was prepared from 20 ml of fresh milk
by centrifugation at 3000 g for 30 min at 15°C. One aliquot (10 ml) of fresh
skimmed milk was immediately analyzed; other aliquots (10 ml each) were frozen
at -20°C and analyzed after a period ranging from 1 to 3 months. Another aliquot
of fresh whole milk was freeze-dried as described in the session “Preparation of
freeze-dried donkey’s milk samples”.
Before to be analyzed, 0.9 g of freeze-dried donkey’s milk was completely
dissolved in 10 ml of distilled water, the reconstituted milk was skimmed as
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Vincenzetti et al.: Donkey’s Milk Nutritional Characteristics
Published by Berkeley Electronic Press, 2011
described before. Whey protein fraction was obtained from skimmed milk by
isoelectric precipitation of the whole caseins at pH 4.6 with 10% (v/v) acetic acid
and centrifugation at 3000g for 10 min in order to obtain the supernatant of whey
proteins.
Donkey’s milk protein profile analysis and whey protein content
determination by reversed-phase chromatography on HPLC (RP-HPLC)
Fresh, frozen, and freeze-dried skimmed donkey’s milk analysis was performed
by a reversed-phase chromatography on HPLC (RP-HPLC; Äkta Purifier Uppsala,
Sweden) as previously described (Vincenzetti et al. 2008). To the samples for RP-
HPLC were added two volumes of clarification buffer (CL buffer: 0.1 M bis-tris,
pH 8.0 containing 8 M urea, 1.3% trisodium citrate, 0.3 % DTT). 100 100 μl of
clarified samples were loaded into RP-HPLC column C4 Prosphere 300Å (5μm,
4.6 mm I.D., 150 mm; Alltech, Waukegan Rd Deerfield, IL) equilibrated in buffer
A (trifluoroacetic acid, TFA/H2O 1:1000 v/v). The elution was achieved by the
following step gradient with buffer B (TFA/H2O/Acetonitrile 1:100:900 v/v): % B
= 0, 10 min; % B = 20, 10 min; % B = 40, 0.1 min; % B = 60, 40 min. The flow
rate was 1 ml/min and fractions of 0.5 ml were collected.
Standard solutions of egg white lysozyme (0.15; 0.25; 0.50; 1.0; 2.0
mg/ml), bovine milk β-lactoglobulin (0.17; 0.30; 0.75; 1.0; 1.5 mg/ml) and bovine
milk α-lactalbumin (0.25; 0.35; 0.50; 0.75; 1.0; 1.5 mg/ml) were prepared in CL
buffer. 100 μl aliquot of each standard was separately loaded on the RP-HPLC
column. The area of each standard peak was measured using the valley-to-valley
integration mode and quantification was achieved by a calibration curve obtained
relating the concentration in micrograms of each standard loaded in the column to
the peak area corresponding to each concentration. The amount of lysozyme, β-
lactoglobulin and α-lactalbumin in fresh, frozen and freeze-dried donkey’s milk
was determined by using the respective calibration curve.
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
analysis
SDS-PAGE analysis was done as described by Laemmli (1970), under reducing
conditions using a 15% acrylamide-bis acrylamide solution and the Mini Protean
III apparatus (Bio-Rad, Hercules, CA; gel size 7x 8cm x 0.75mm). The markers
used were Bio-Rad molecular weight standards, low range. Electrophoresis was
performed at 4 °C with a constant voltage of 200 V. The proteins were visualized
on the gel by Coomassie Blue staining (0.1% Coomassie Brilliant Blue R250 in
50% methanol and 10% acetic acid).
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Probiotic strains
The Lactobacillus rhamnosus IMC 501® and Lactobacillus paracasei IMC 502®
probiotic strains (Verdenelli et al. 2009) were obtained from Synbiotec S.r.l.,
Camerino, Italy. The strains were supplied as freeze-dried powder with a
concentration of 1 x 1011 CFU/g.
Preparation of freeze-dried donkey’s milk samples
12 mg of the probiotic strains mixture (1:1) were added to 12 mL of raw donkey’s
milk, gaining a final concentration of 1 g/L. Raw donkey’s milk not enriched with
probiotics was used as control sample (12 mL). Sample and control were frozen at
−80 °C in a static state for 24 h. Successively, they were dried in a Zirbus freeze
dryer (Zirbus Vaco 2, Bad Grund, DE) with a condenser temperature of −50 °C
and a chamber pressure P < 0.08 mbar for 48 h.
Probiotic viability during storage
Freeze-dried sample and control were stored in portion-size sample sachets and
under vacuum. They were placed at 4 °C to evaluate their stability. The viability
of the freeze-dried samples was performed through plate count technique at
production day and over 2 months of storage using de man, Rogosa, Sharpe agar
(MRS) (Oxoid LTD., Basingstoke, Hampshire, England) supplied with
vancomycine (12 mg/L) (Sigma-Aldrich S.r.l., Milan, Italy) as medium. At each
sampling time (production day, 15, 30, 45, 60 days) 0.25 g of freeze-dried
samples were analysed.
Other analytical procedures
Protein concentration was determined by the Bradford protein assay (Bradford
1976). Vitamin C content determination was performed using the kit ENZYPLUS
EZA 941+ L-Ascorbic Acid (Raisio Diagnostics) in which is exploited the ability
of L-ascorbic acid to reduce the tetrazolium salt MTT [3-(4,5dimethylthiazolyl-2)-
2,5-diphenyltetrazolium bromide] in the presence of the electron carrier PMS (5-
methylphenazinium methosulfate) at acidic pH to a formazan. The MTT-formazan
is determined by means of its light absorbance in the visible range at 578 nm.
Lysozyme activity was performed by turbidimetric assay according to the
method of Jenzano et al. (1986) by using a Beckman DU 640 UV-VIS
spectrophotometer as described previously (Polidori and Vincenzetti 2010). This
assay is based on the use of Micrococcus lysodeikticus as substrate: lysozyme
catalyzes the hydrolysis of glycoside bonds of muramic acids of the bacterial cell
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Vincenzetti et al.: Donkey’s Milk Nutritional Characteristics
Published by Berkeley Electronic Press, 2011
walls that results in the lyses of the bacterial cells and clarification of the reaction
medium. The substrate was prepared just before use under sterile conditions by
mixing gently 30 mg of the freeze-dried cells Micrococcus lysodeikticus with 100
ml of sterile 0.1 M sodium phosphate pH 7.0 and incubated 1 hour at 25°C. The
reaction mixture contained 10-100 μl of skimmed milk and substrate up to 1 ml.
The time-depended decrease in optical density at 490 nm was monitored for 6 min
and were made at least 5 determination for each sample. The control solution
contained only 0.1M sodium phosphate pH 7.0 buffer. One unit was defined as the
amount of lysozyme that caused the decrease of 0.001 unit of absorbance at 490
nm per minute at 25°C. Standard lysozyme solutions: 2.0 mg/ml; 4.0 mg/ml; 6.0
mg/ml; 8.0 mg/ml in 0.1M sodium phosphate pH 7.0 buffer were used to test the
linearity of the turbidimetric assay.
Statistical analysis
Data were analysed by the method of least squares using the general linear model
procedures of SAS (2001) and results were expressed as least square means.
Significant differences between means were indicated when P<0.05.
RESULTS AND DISCUSSION
Donkey’s milk protein content determination, vitamin C quantification and
lysozyme activity
In Table 1 are resumed the results on the effects of lyophilization treatment on
some donkey’s milk nutritional characteristics and the comparison with fresh and
frozen milk.
The α-lactalbumin, β-lactoglobulin and the total caseins content resulted
to be not statistically different in fresh and in freeze-dried donkey’s milk, on the
contrary, when milk was preserved at -20°C was observed a significant decrement
of the caseins and total proteins content. After one and two months of strage at –
20°C, total proteins content significantly (P<0.05) decreased, and this negative
trend was increased at three months (P<0.01). Total caseins content showed a
significant (P<0.05) decrease after one month of storage at –20°C, while after two
and three months the total amount of caseins significantly (P<0.01) decreased
again. Lysozyme amount and enzymatic activity were not affected by the different
storage conditions analyzed: α-lactalbumin and β-lactoglobulin content resulted
almost stable in frozen milk until three months.
L-Ascorbic acid (vitamin C) can be used as a quality indicator in the
production of several foods and derivatives such as wine, beer, milk, soft drinks
and fruit juices. This vitamin plays a rule as anti-oxidant and free radical
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scavenger; for this reason its quantitative determination is important. In this work
the content of vitamin C has been determined for the first time in fresh donkey’s
milk and also the amount of this vitamin was evaluated after lyophilization and
frozen treatment. The results, shown in table 1 indicated that the fresh donkey’s
milk contains 57 mg of vitamin C per liter. This value is very similar to that one
obtained in human milk by Hoppu et al. (2004), and is absolutely higher
compared with that one determined on cow’s milk, 15 mg per liter (Morrissey,
2003). The lyophilization process did not significantly affected the amount of
vitamin C in this milk, while vitamin C content significantly decreased (P<0.01)
after one, two and three months of storage at -20°C.
Table 1: Donkey’s milk protein content determination, vitamin c quantification
and lysozyme activity on fresh, lyophilized and frozen milk (1, 2 and 3 months at
-20°c).
Fresh Lyophilized Frozen
(months at - 20°C)
1 2 3
Total proteins (mg/ml) 8.68a8.24a 7.41
b
7.28
b
6.15c
Total caseins (mg/ml) 1.43a1.61a 0.93
b
0.66
b
0.36c
Lysozime (mg/ml) 2.18 1.84 2.02 1.94 2.22
Lysozime activity
(U/ml)
0.62 0.69 0.69 0.72 0.69
α-lactalbumin (mg/ml) 2.40a1.89
b
2.26a 2.27a 2.40a
β-lactoglobulin
(mg/ml)
5.54 5.47 5.15 5.24 5.02
Vitamin C (mg/L) 57.0a51.0
b
45.0c 41.0c 35.5c
Different letters on the same row indicate a statistical difference: b: P<0.05; c:
P<0.01)
Donkey’s milk protein profile analysis in the different storage conditions
In figure 1 is shown the chromatographic protein profile of RP-HPLC followed by
15% SDS-PAGE performed on fresh, freeze-dried and frozen (3 months at -20°C)
donkey’s milk. The protein profile obtained by RP-HPLC resulted similar in the
fresh and in the freeze dried donkey’s milk. 15% SDS-PAGE analysis (data not
shown) of each peak separated from each RP-HPLC (figure 1) allowed to the
identification of the main protein components in donkey’s milk at different
storage conditions: peak 1 that correspond to lysozyme with a molecular weight of
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Vincenzetti et al.: Donkey’s Milk Nutritional Characteristics
Published by Berkeley Electronic Press, 2011
14.60 kDa, peaks 2, and 4 corresponds to the casein fractions (molecular weight
ranging from 44.0 to 29.0 kDa) that are lowest in frozen milk with respect to fresh
and freeze-dried milk. Peak 3 corresponds to α-lactalbumin with a molecular
weight of 14.12 kDa, whereas the peak 5 is the β-lactoglobulin with a molecular
weight of 22.40 kDa.
Figure 1. Reversed-phase HPLC of fresh, lyophilized and frozen donkey milk. the
chromatographic course has been performed as described under materials and
methods session.
Probiotic viability during storage
Probiotics are live microorganisms which when administered in adequate amounts
confer a health benefit on the host (WHO, 2001). They are non-pathogenic
microorganisms that are able to reach the colon alive, where they exert a positive
effect on the health of the host. Recently, several studies focused on the
importance of the intestinal microflora and in particular on the effects of
probiotics on acute infectious gastroenteritis especially in infants. The protective
effect of probiotic is due mainly to a direct antagonism against pathogenic strains
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through a competitive inhibition mechanism of adhesion to the gastrointestinal
mucosa and to an immune stimulation (Salvatore et al. 2007). In particular, in
infants the most significant demonstrated effects of probiotics include prevention
and treatment of antibiotic-associated diarrhea and rotavirus diarrhea and allergy
prevention (Salminen and Isolauri 2006).
During storage (figure 2), the viability of both probiotic strains in freeze-
dried donkey’s milk powder was unchanged for the two months following the
production day, maintaining a count of about 2 x 108 CFU/g. The analyses of
control samples revealed an absence of bacterial count. Freeze-drying is a
common method used to preserve foods from spoilage. Nowadays this technique
is extensively investigated with the aim to incorporate probiotics in food products
for the preparation of functional foods (Savini et al. 2010). Moreover,,viability
appears to be an important factor in probiotics to facilitate health effects. Some
authors reported that only viable probiotics in extremely sensitive infants were
able to alleviate symptoms of atopic dermatitis (Kirjavainen et al. 2003; Isolauri et
al. 2000).
Figure 2. Probiotic viability during storage of donkey milk by freeze-drying
technique.
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Vincenzetti et al.: Donkey’s Milk Nutritional Characteristics
Published by Berkeley Electronic Press, 2011
The experimental results pertaining to survival of freeze-dried donkey’s
milk containing L. rhamnosus and L. paracasei evidence the optimal
cryoprotection carried out from the donkey’s milk during lyophilisation and when
stored at refrigerated temperature. The protective affect is likely due to the high
content of lactose (Guo et al. 2007), which is already known to be efficient in
protecting biological systems from freezing and lyophilisation injuries (Zhao and
Zhang 2005, Pehkonen et al. 2008). The high content of lysozyme in donkey’s
milk seems did not affect the probiotic strains viability during storage according
to Coppola et al. (2002); this results confirmed the possibility of producing a
probiotic infant formula with favorable beneficial properties using donkey’s milk
as raw material, as demonstrated also by Chiavari et al. (2005).
CONCLUSION
Many clinical studies indicated donkey’s milk as a valid substitute of cow’s milk
for feeding allergic children, because of the structural similarities between human
and donkey’s milk proteins. The treatment of lyophilization of donkey’s milk
demonstrated that the nutritional characteristics of this product remained basically
unchanged compared with fresh milk; as a consequence, lyophilized donkey’s
milk could be evaluated in further studies as a new dietetic food for infant
nutrition in replacing of breast milk. The high lysozyme content in donkey’s milk
does not create a negative interaction with the possible supplementation with
probiotic strains, giving therefore the opportunity of using donkey’s milk for the
production of probiotic beverages. Due to the recent interest in use of donkey’s
milk for the treatment of CMPA, the knowledge of the effects of lyophilization of
this product and the selection of other bacterial strains with probiotic properties
can be deepened in order to investigate on other bionutritional parameters after a
treatment that can help in supplying donkey’s milk on the market allover the year.
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