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Effects of Lyophilization and Use of Probiotics on Donkey's Milk Nutritional Characteristics Effects of Lyophilization and Use of Probiotics on Donkey's Milk Nutritional Characteristics

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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.
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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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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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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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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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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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... Donkey milk casein fractionation is obtained from mainly αs 1 and βcaseins. The fractionation of donkey milk casein is subjected to various studies like 1D and 2D electrophoresis, structural mass spectrophotometry analysis and reversed phase-High Performance Liquid Chromatography (Vincenzetti, et al., 2012, Chianese, et al., 2010. Furthermore donkey milk contains αs 2 -casein and k-casein in a minor amount (Bertino et al., 2010, Chianese, et al., 2010). ...
... Low amount of total plate count in donkey milk is mainly because the donkey milk hasample amount of natural antibacterial component like lactoperoxidase and lactoferrin. Lactoperoxidase content in donkey milk is about 0.11 mg/mL (Shin et al., 2001) while the lactoferrin content in donkey milk is about 0.080 (Vincenzetti et al., 2012). Donkey milk contains LAB between 1.0 to 4.2 log cfu/ml, but very less species are isolated and identified (Chiavari et al., 2005, Coppola et al., 2002, Zhang et al., 2008, Carminati et al., 2014. ...
Book
Full-text available
... The lack of vitamin B12 in equid milk compared to ruminant milk could be explained by the different digestive systems among these two species, and vitamin B12 is synthesized by the microorganisms of the digestive tract. In addition, the vitamin C content in DM is very similar to that in breast milk but higher than that in cow's milk (Table 4) [2,49,55,56]. The vitamin A content in DM (58 µg/100 mL) is slightly lower compared to that in breast milk (60 µg/100 mL) [57], while the vitamin D content in DM is higher than the values found in the milk of many other mammals and in breast milk [45]. ...
... Vitamin content in donkey milk[2,49,56,57]. ...
Article
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Donkey milk (DM) has been known in the world for 5000 years for its benefits for human nutrition and health. Nowadays, DM has become more and more attractive as a commercial product. DM contains several physiologically functional components, including high-quality whey proteins, vitamins, important minerals, unsaturated fatty acid and bioactive components. Therefore, it is not only consumed as food but also as a remedy. The average daily milk yield of a female donkey over the entire lactation season was 1.57 ± 1.12 kg/day and fluctuated between 0.20 and 6.00 kg/day. Average milk concentrations (±SD) of fat, protein, lactose, total solids and ash in DM were 0.63 ± 0.41%, 1.71 ± 0.24%, 6.34 ± 0.37%, 9.11 ± 0.95% and 0.39 ± 0.04%, respectively. Interestingly, DM is similar in composition to mare’s milk, and both are similar to mother’s milk. The anatomical and morphological properties of the mammary gland of the female donkey are special and can be compared with those of mare udders. However, the cistern cavity of the mammary gland of female donkeys is characterized by the presence of multiple pockets that open directly into the teat, instead of a single cistern cavity. Therefore, the mammary gland capacity in donkey mare is low and milking technique and routine are of most importance. So far there is no special milking machine for female donkeys and mares. The milking machines used nowadays were initially designed for smaller sheep and goat udders. The company Siliconform, Germany, has set itself the task of developing an optimized milking machine for donkey mares, which is adapted to the anatomical and morphological properties of the donkey mammary gland. Furthermore, it should achieve a physiologically ideal milking process meeting high animal welfare standards for increased milk production with high quality standards.
... Donkey milk casein fractionation is obtained from mainly αs 1 and βcaseins.The fractionation of donkey milk casein is subjected to various studies like one-dimensional electrophoresis, two-dimensional electrophoresis, structural MS analysis, andreversed phase-HPLC (Vincenzetti, et al., 2012, Chianese, et al., 2010. Furthermore donkey milk contains αs 2 -casein and k-casein in a minor amount (Bertino et al., 2010, Chianese, et al., 2010). ...
... Low amount of total plate count in donkey milk is mainly because the donkey milk hasample amount of natural antibacterial component like lactoperoxidase and lactoferrin. Lactoperoxidase content in donkey milk is about 0.11 mg/mL (Shin et al., 2001) while the lactoferrin content in donkey milk is about 0.080 (Vincenzetti et al., 2012). Donkey milk contains LAB between 1.0 to 4.2 log cfu/ml, but very less species are isolated and identified (Chiavari et al., 2005, Coppola et al., 2002, Zhang et al., 2008, Carminati et al., 2014. ...
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Full-text available
... Moreover, some unique nutritional qualities as rich source of whey proteins approx. 35-50% of the nitrogen content [6], high lysozyme concentration in comparison to goat, ewes, cow and other equines [7] and low concentration of fat approx. 0.28% to 1.82% (Carroccio et al. [8]) are also reported in donkey milk. ...
... Vitamin C 5600 μg/100 ml 5700 μg/100 ml 1300 μg/100 ml 4600 μg/100 ml 1500 μg/100 ml Vincenzetti et al., [7], Moltó-Puigmartí et al., [41] & Keszycka et al., [32] and Balthazar et al., [33] pH 7.0-7.5 7.0-7.2 6.6 6.6 6.6-6. ...
... Yağsız sütün, -80C'de en iyi koruyucu ajan olduğu bildirilmiĢtir [175,177] . Geleneksel olarak laktoz ve yağsız süt gibi süt bazlı ürünler, bakterilerin korunmasında kriyo-koruyucu olarak kullanılmaktadır [178,179] . ...
Chapter
Bu bölümde tüm bu gereklilikler dikkate alınarak hem temel probiyotik özellikler, hem de endüstriyel özellikler açısından probiyotik suşlarda aranması gereken kriterler açıklanmıştır.
... The average vitamin C content is 5,700 mg/100 mL in donkey milk which comparable to that of human milk, which is about 5,600 mg/100 mL. The average vitamin C content in cow's milk is about 1,500 mg/100 mL [42,43]. A higher vitamin D content (2.3 mg/100 mL ± 0.86) (about 92 UI) was reported in Amiata donkey milk. ...
... Salimei and colleagues (Salimei et al. 2004) showed that the average concentration of lysozyme in DM is three times higher than in human milk, while this component is absent in the milk of cows, ewes and goats (Vincenzetti et al. 2007). Lysozyme in DM ranges from 0.67 to 3.74 g/L and maintains the same high percentage over the total protein during 150 days of lactation (Guo et al. 2007, Vincenzetti et al. 2011, Šarić et al. 2012, Šarić et al. 2014. The interaction between lactoferrin and the lipopolysaccharidic layer (LPS) causes disruption of the outer membrane. ...
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Gram-positive foodborne pathogens such as Listeria monocytogenes and Staphylococcus aureus can grow in a wide variety of foods, including raw milk. The aim of the study was to compare the growth of L. monocytogenes and S. aureus inoculated in donkey and cow samples of raw milk during a storage time of 11 days at 8 °C. Moreover, the study aimed to evaluate the influence of lactic acid bacteria (LAB) content on the growth of the two microbiological populations considered. LAB content was lower in raw donkey milk than in raw cow's milk during the entire analyses; on the other hand, pH levels were higher in the donkey milk rather than in the cow's milk, although both values showed a decrease at the day 11. S. aureus showed no significant differences in the two types of milk. From day 0 to 11, L. monocytogenes increased from 3.68 ± 0.02 log CFU/mL to 6.31 ± 0.07 log CFU/mL and from 3.64 ± 0.04 log CFU/mL to 4.59 ± 1.04 log CFU/mL, in donkey milk and in cow's milk, respectively. Our results showed that donkey milk is a more favourable matrix to support the growth of L. monocytogenes than cow's milk.
... The donkey milk is enrich in protein, minerals, essential fats, bioactive enzymes and various growth factors like ribofl avin, vitamin D etc. provide natural nourishment to skin and toned it. The donkey milk naturally contain antibacterial compound such as lysozyme and lactoferrin inhibit the growth of pathogenic bacteria on skin and reduce the rate of skin infection [33,34]. Due to these properties the donkey milk formulated with some chemical may used to treat acne, psoriasis, eczema and other related skin infection. ...
Article
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The moisture sorption isotherms of donkey milk powder were studied at 20, 25 and 30 °C for 0.22 to 0.93 aw through gravimetric method. GAB, Peleg, Smith and Oswin mathematical models were fitted to experimental data. Equilibrium moisture content of the sample increased from 0.0499 to 0.3255, 0.0447 to 0.2904, 0.0426 to 0.2618 kg water. kg‐1 dry solid at 20, 25 and 30 °C with increasing aw from 0.22 to 0.93. Isotherms curves showed type II behaviour. Monolayer moisture content decreased from0.0373 to 0.0301 g.100g‐1 solids at 20 to 30 °C. The net isosteric heat of sorption decreased from 98.55 to 0.74 kJ.mol‐1 with increased moisture content from 2 to 20% (d.b.). The adsorption isotherm curves suggest that the sample requires more attention when handled in RH higher than 24, 33 and 42% at 20, 25 and 30 °C.
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Background: Whole milk is a good source of all the nutrients, and it also contains a sufficient number of vitamins to permit regular the growth of the neonate. Dairy cow milk can create allergy in infants less than 12 months old because of the high caseins and β-lactoglobulin content. In these circumstances, donkey milk can represent a good replacement for dairy cows' milk in children affected by Cow Milk Protein Allergy (CMPA) because of its close chemical composition with human milk, mainly due to its low protein and low mineral content. Milk vitamin content is highly variable among mammalian species and it is strictly correlated with the vitamin status and the diet administered to the mother. Fat-soluble vitamins content in donkey milk is, on average, lower compared to ruminants' milk, while vitamin C content determined in donkey milk is higher compared to dairy cows' milk, showing a great similarity with human milk. In donkey milk, the content of vitamins of the B-complex such as thiamine, riboflavin, niacin, pyridoxine, and folic acid is higher compared to human milk. The use of donkey milk as a new functional food must be further evaluated in interdisciplinary clinical trials in which pediatricians, dietitians, and food scientists must be involved to deepen the knowledge about the positive health impact of donkey milk in different sensitive people, especially children and the elderly.
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Milk is one of the most common causes of food allergies among children under one year of age. No specific therapy exists for this allergy, and thus the only feasible response is to avoid assumption of milk and derived products. Studies conducted on the serum of children with hypersensitivity to milk have shown that caseins are the proteins with the greater allergenic potential. However, in some cases, children have also shown hypersensitivity to the -lactoglobulines and to the -lactalbumins. When food intolerance is diagnosed in an infant, it is often necessary to impose a period of total parenteral feeding, followed by breast feeding, considered the most correct method of re-feeding; when human milk cannot be given, alternative food sources must be sought. Clinical studies have demonstrated that donkey milk could substitute breast feeding in infants affected by severe Ig-E mediated milk allergies. In these subjects, donkey milk is not only useful, but also safer than other types of milk. In fact donkey milk composition in lipids (high levels of linoleic and linolenic acid) and proteins (low caseins content) is very close to human milk. In our studies on donkey milk we tried to deepen the knowledge about this product especially regarding the protein fractions. Among caseins, we identified mainly  S1 -and -caseins, other types of caseins in such as -and -caseins were not found probably because of their low amount. Lysozyme content in donkey milk resulted to be very high (mean value 1.0 mg/ml) if compared to bovine (traces), caprine (traces) and human milk and varied at different stages of lactation. The high lysozyme content of donkey milk may be responsible of the low bacterial count reported in literature and also makes this milk suitable to prevent intestine infection to infants.
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Aim was to investigate the efficacy of heat treatment on microbiological and biochemical quality of ass's milk and its effects on the growth of 4 different strains of Lac-tobacillus rhamnosus. Milk samples were obtained from 6 machine-milked asses (Martina Franca breed). Low bacterial counts (about 4x10 4 CFU/mL) were observed in raw milk samples likely due to high levels of lysozyme. The possible role of lysozyme was also sug-gested by stable pH values during storage for 15 days. Moreover, ass's milk demonstrated to be a good growth medium for potentially probiotic strains of L. rhamnosus since pH ran-ged between 3.67 and 3.85 for all the tested strains after a 48-hour period of incubation. Values were stable up to the end of the trials. Results evidenced that ass's milk is indeed an excellent candidate for probiotic beverages.
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A detailed kinetic study of alkaline phosphatase, lactoperoxidase and temperature indicators for controlling the heat processing of milk. The heat inactivation or denaturation of alkaline phosphatase, lactoperoxidase and -lactoglobulin we found z values of 7·9 deg C (D75 °C = 49·9 min) in the temperature range 70–80 °C and 24·2 deg C (D85 °C = 3·53 min) in the range 83-95 °C. Dref and z were evaluated under dynamic temperature conditions. To estimate the statistical accuracy of the parameters, 90% joint confidence regions were constructed.
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The human intestine is colonized by a large number of microorganisms that inhabit the intestinal tract and support a variety of physiological functions. The stepwise microbial colonization of the intestine begins at birth and continues during the early phases of life to form an intestinal microbiota that is different for each individual subject. This process facilitates the formation of a physical and immunologic barrier between the host and the environment, helping the gastrointestinal tract maintain a disease-free state. Probiotics are viable microbial food supplements that have a beneficial impact on human health. Health-promoting properties have been demonstrated for specific probiotic products. Scientific data are accumulating on these properties, especially in infants; the most significant effects include prevention and treatment of antibiotic-associated diarrhea and rotavirus diarrhea and allergy prevention. Bifidobacteria appear to be the most promising probiotic candidates, followed by defined lactic acid bacteria, which favor specific healthy bifidobacterial growth and species composition. Because viability appears to be important, probiotic properties also should be emphasized to meet this criterion. For future probiotics, the most important requirements include a demonstrated clinical benefit supported by mechanistic understanding of the effect on target population microbiota and immune functions. Genomic information and improved knowledge of microbiotic composition and its aberrancies should serve as a basis for selecting new probiotics for use in specific infant populations.
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Through our current knowledge of milk protein structure and polymorphism, and the molecular biology of the relevant genes, we show how DNA technology can contribute to improving milk protein quality.Characterization of the goat αs1-casein gene polymorphism and the establishment of its overall structural organization have provided information necessary for developing an allele specific typing procedure. Relying on the polymerase chain reaction technique, this procedure has proven to be a potential tool for selection, which still remains the most efficient way to improve livestock production traits. Alternatively, recent progress in gene transfer technology makes feasible generation of transgenetic dairy animals, producing milk whose composition would be modified to alter its physicochemical and nutritional properties for specific purposes such as producing “humanized” milk in the mammary gland of ruminants. Due to the lack of embryo-derived stem cells from large domestic animals, we can only anticipate that gene targeting, which seems to be the method of choice to direct specific mutations within any coding or regulating region of endogenous genes, will undoubtedly result in modifying livestock genomes.
Article
Summary Thermal indicators in milk, which had been subjected to one of the six industrial processes of thermization, pasteurization, direct and indirect UHT-sterilization, pre-sterilization and in-bottle sterilization, were studied. The following three indices of heat damage were analyzed by high performance liquid chromatography (HPLC), hydroxymethylfurfural (HMF), lactulose and acid-soluble β-lactoglobulin(β-LG). Average amounts found were 1710 mg/l of β-LG and 2.49 μmol/l of HMF in pasteurized milk. In UHT milk, the amounts for direct and indirect processes were 389 and 322 mg/l of β-LG, 12.0 and 250 mg/l of lactulose and 5.6 and 8.7 μmol/l of HMF. In sterilized milk the amounts were 1120 mg/l of lactulose and 22 μmol/l of HMF, without any detectable presence of undenatured whey proteins. On the basis of the time/temperature profiles, a sterilization factor, expressed as seconds, was defined for each thermal treatment. By applying discriminant analysis each industrial process could be classified independently at the 95% confidence level (pasteurization, UHT-treatment and in-bottle sterilization), but direct-UHT treated milk could not be discriminated from indirect-UHT milk, nor thermized milk from raw bulk milk. The simultaneous application of several heat-induced parameters improves the classification of industrial processed milks, and is therefore a useful tool for optimization of the processing conditions.