_

Food Science and Technology Research, 21 (4), 573 581, 2015

Copyright © 2015, Japanese Society for Food Science and Technology
doi: 10.3136/fstr.21.573

http://www.jsfst.or.jp

Original paper

Effects of Sterilization Process on the Physicochemical and Nutritional

Properties of Liquid Enteral Formula

Yasuhiro Takeda1, Masayuki Shimada2*, Yoshihiko Ushida2, Hitoshi Saito2, Hiroshi Iwamoto1 and
Teiichiro Okawa2

1
Nutritional Science Institute, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 252-8583, Japan
2
Food Research & Development Institute, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 252-8583, Japan

Received December 26, 2014 ; Accepted March 26, 2015

Ultra-high temperature (UHT) and retort processes are widely applied in the sterilization of liquid enteral
formulae. This study investigated the effects of sterilization process on the physicochemical and nutritional
properties of a liquid enteral formula. An ingredient mixture was sterilized by either UHT processing at 150℃ for 5 s,
or retort processing at 121℃ for 10 min (retort A) or 121℃ for 40 min (retort B). In vitro assays showed less protein
modification in the UHT formula than in either retort formula. In vivo assays also demonstrated that net protein
utilization was greatest with the UHT formula, and cecal nitrogen and ammonia concentrations in rats fed the UHT
formula were lower than in rats fed either retort formula. These results indicate that the sterilization process affects
the protein quality of the formula as well as the intestinal environment of rats fed the formula.

Keywords: enteral formula, sterilization, glycation, protein quality, intestinal environment

Introduction In UHT processing, the enteral formula is heated at a high

In recent years, the number of elderly individuals requiring temperature for a few seconds and packaged under aseptic
nutritional care has increased in parallel with the world’s aging conditions. In contrast, in retort processing, the enteral formula is
population, including in Japan. Various enteral formula products packaged in a thermally resistant container and heated at a high
have been developed to maintain an adequate nutritional status in temperature for minutes. The time required for retort sterilization is
the elderly. Enteral formulae are also used as a nutritional source clearly much longer than for UHT sterilization. Generally, the
for patients with various disorders. Therefore, these products must temperature required for retort sterilization is lower than for UHT
have a high standard of hygienic quality. To ensure a high level of sterilization.
microbial safety in enteral formula products, sterilization is Heat treatment stimulates the Maillard reaction in milk and
generally performed during the production process using widely dairy products, which is related to the heating temperature and
applied methods such as ultra-high temperature (UHT) or retort duration (van Boekel, 1998). Because the amino group of amino
processing. acids, especially lysine, is blocked by the reducing sugar in the

Abbreviations
UHT; ultra-high temperature, AGEs; advanced glycation end products, OPA; o-Phthalaldehyde, CML; Carboxymethyllysine, SDS; sodium
dodecyl sulfate, SDS-PAGE; SDS-polyacrylamide gel electrophoresis, TCA; trichloroacetic acid, FER; food efficiency ratio, TD; true
digestibility, BV; biological value, NPU; net protein utilization

*To whom correspondence should be addressed. E-mail: m-shimada@morinagamilk.co.jp

574 Y. Takeda. et al.

Maillard reaction (Rufián-Henares et al., 2006; van Boekel, 1998), Liquid enteral formulae Sodium caseinate, as the sole protein
it is thought that heat treatment affects protein quality (Desrosiers source, was mixed with sugar, dextrin, cellulose, vegetable oil, fish
and Savoie, 1991; Kilshaw et al., 1982; Rutherfurd and Moughan, oil, minerals, and vitamins. These ingredients and their ratios were
2005; Sarwar et al., 1988). based on the formulation of our commercial liquid enteral product.
In fact, Sarwar et al. (1989) investigated the protein quality of The mixture was subjected to three sterilization processes: a UHT
commercial infant formulae manufactured by different production process (UHT formula) and two different retort processes (retort A
processes in a rat study, and found that the digestibility and protein and retort B formulae), at the pilot plant of Morinaga Milk Industry
efficiency ratio of the liquid formula were lower than those of the Co., Ltd. Each sterilization condition was based on the
powder. In contrast, Sarriá et al. (2000) investigated the protein manufacturer’s commercial product conditions (Table 1). The F
quality of various commercial formulae, including a powdered value is used in the food industry to evaluate the intensity of the
form, an in-bottle-sterilized form and a UHT form, and reported no sterilization (Ramaswamy and Marcotte, 2006). The F value of the
remarkable differences in product digestibility and protein retort B process was designed to be similar to that of the UHT
utilization in rats. In these studies, commercial formula products process. The F value of the retort A process was set at a lower
were used; thus, it is likely that the inconsistent results can be value than the UHT and retort B processes. These test formulae
attributed to differences in ingredients and composition. were kept at 4℃ until use. We prepared a non-protein formula
Additionally, the sterilization conditions employed were not (formula without protein) containing the same ingredients as the
described in detail. formula but omitting sodium caseinate. The dextrin content of the
In a preliminary study, we found differences in the sensory non-protein formula was increased to ensure that the total caloric
properties of UHT and retort treated liquid enteral formulae, which value was the same as the other test formulae. The non-protein
appeared to be related to the Maillard reaction (data not shown). formula was kept frozen until use, and was not subjected to
The aim of this study was to investigate the influence of sterilization treatment. The compositions of test formulae are
sterilization process on dietary protein properties such as shown in Table 2. Some vitamins are heat labile and might be
physicochemical characteristics and nutritional value. To destroyed by heat treatment (Asadullah et al., 2010; Haddad and
investigate the direct effect of sterilization process on the protein Loewenstein, 1983); therefore, to prevent vitamin deficiency, a
quality of liquid enteral formulae, test formulae were prepared vitamin mixture for rodent diets (AIN-93G vitamin mix; Oriental
from the same ingredient mixture, i.e., the same ingredient mixture Yeast Co., Ltd., Tokyo, Japan) was added to each of the four test
was subjected either to UHT sterilization (150℃, 5 s) or two kinds formulae used in the animal assay. In this study, we employed
of retort processes (retort A: 121℃, 10 min; retort B: 121℃, 40 identical test formulae in both the in vitro and in vivo assays.
min), and the protein quality was evaluated both in vitro and in In vitro studies
vivo using rats. Colorimetry Color determination of the test formulae was
In addition, we investigated the intestinal environment of rats performed using the Spectro Color Meter SE2000 (Nippon
fed the test enteral formulae. Dietary protein is digested and Denshoku Industries Co., Ltd., Tokyo, Japan) according to the Lab
absorbed in the upper intestine. However, any protein not fully color solid (Seiquer et al., 2010), where “L” represents the black to
digested may reach the lower intestine and act as a substrate for white component as the luminosity, while “a” represents the +red
intestinal bacteria, resulting in its conversion to metabolites such as to _green component and “b” represents the +yellow to _blue
ammonia. Regarding the relationship between the human intestinal component as the chromaticity coordinates.
environment and the amount of dietary protein intake, Cummings Furosine Furosine is used as an index of early Maillard
et al. (1979) reported that subjects consuming an experimental diet reaction products. The amount of furosine in the test formulae was
with excessive protein showed elevated fecal ammonia determined using high-performance liquid chromatography
concentrations. Therefore, we hypothesized that the sterilization (Shimadzu Corporation, Kyoto, Japan) with a Mightysil RP-18GP
process may affect digestibility, and that the undigested protein column (250 × 4.6 mm) (Kanto Chemical Co., Inc., Tokyo, Japan),
may reach the lower intestine and alter the intestinal environment. following hydrolysis of the test formulae with 10 M HCl at 110℃
Thus, the cecal nitrogen and ammonia concentrations of rats fed for 10 h in evacuated sealed tubes, according to the methods of
the test enteral formulae were investigated.
Table 1 . Heat treatments of the test formulae .
Materials and Methods
Sterilization Holding temperature and time F value
Materials Furosine (ε-N-(furoylmethyl)-L-lysine) was
UHT 150℃ for 5 s 80
purchased from Neosystem Laboratories (Strasbourg, France).
Retort A 121℃ for 10 min 15
o-Phthalaldehyde (OPA) was from Tokyo Chemical Industry Co.,
Retort B 121℃ for 40 min 70
Ltd. (Tokyo, Japan). Porcine pepsin and pancreatin were from
Sigma Aldrich Co., LLC. (St. Louis, MO, USA). F value is an index of the intensity of the heat treatment .
Effects of Sterilization on Liquid Enteral Formula Protein Quality 575

Table 2 . Composition of the test formulae .

Component UHT formula Non-protein formula

Retort A formula
Retort B formula
Protein (g/100 mL) 4.0 0
Fat (g/100 mL) 3.0 3.0
Carbohydrate (g/100 mL) 14 . 3 18 . 3
Dietary fiber (g/100 mL) 0.4 0.4
Ash (g/100 mL) 0.6 0.6
Moisture (g/100 mL) 85 . 0 85 . 0
Calories (kcal/100 mL) 100 100

Delgado et al. (1992) and Guerra-Hernandez and Corzo (1996). Japan SLC, Inc. (Shizuoka, Japan), and were housed individually
Carboxymethyllysine (CML) CML is a form of advanced in metabolic cages in an air conditioned room with a 12 h light/
glycation end-products (AGE) produced in the later stages of the dark cycle. During the 1 week acclimation, all rats were allowed
Maillard reaction. The amounts in the test formulae were quantified free access to the UHT formula. The UHT formula was employed
using a competitive ELISA kit (CycLex Co., Ltd., Nagano, Japan). to accustomize the rats to the liquid test formula.
Available lysine The available lysine (unmodified lysine) The animal experiment was approved by the animal
content was determined based on the method of Goodno et al. experimentation committee of Morinaga Milk Industry Co., Ltd.,
(1981). Briefly, 500 µL of the test formula was added to 500 µL of and was performed in accordance with the company’s regulations
sodium dodecyl sulfate (SDS) (12% w/w) solution and kept at 4℃ and the various laws and guidelines for animal experimentation in
overnight. This solution was sonicated and mixed with freshly Japan.
prepared OPA reagent in the wells of a microplate for 2 min, and Animal assay The animal assay was performed to investigate
the relative fluorescence was determined immediately at excitation the protein quality of the test formulae in rats, based on the
and emission wavelengths of 340 and 455 nm, respectively. Casein, methods reported previously (AOAC, 2005; van Dael et al., 2005).
prepared in our institute from raw milk, was used as a reference After acclimation, rats were randomly divided into four groups
protein. of eight to ten animals and assigned to the following four diet
SDS-polyacrylamide gel electrophoresis (SDS-PAGE) SDS- groups: UHT formula (UHT group), retort A formula (retort A
PAGE was performed according to the method of Laemmli (1970) group), retort B formula (retort B group), and non-protein formula
on a 5 _ 14% polyacrylamide gradient gel (TEFCO, Tokyo, Japan) (non-protein group). The rats were fed their respective test formula
under reducing conditions. The gel was stained using Coomassie ad libitum during the 8 day test period, and body weight and food
Brilliant Blue R250 (Sigma Chemical Co.). consumption for all animals were recorded daily. During the final 3
In vitro digestion In vitro digestion of the test formulae was days of the test period, urine and feces were collected daily from
performed according to the method of Seiquer et al. (2010). Each individual rats and stored at _ 80℃ until analysis. The feces
test formula was adjusted to pH 2.0 with the addition of 6 M HCl collected were lyophilized, weighed and ground, and the nitrogen
for peptic hydrolysis. The formulae were incubated at 37℃ for 120 contents of the dry feces and urine were determined. The nitrogen
min with 0.06% (w/v) pepsin. Following peptic hydrolysis, the contents of the test formulae were also determined. The data from
formulae were adjusted to pH 7.0 using 5 M NaOH and incubated the non-protein group were used to correct for the influence of non-
at 37℃ for 120 min with 0.05% (w/v) pancreatin. Immediately dietary nitrogen from, for example, gut bacteria and sloughed
following incubation, the formulae were cooled in an ice bath. The intestinal mucosal cells.
degree of digestion was determined according to the method of Blood samples were obtained from the abdominal vein of rats
Rudloff and Lönnerdal (1992). Following digestion, trichloroacetic at the end of the test period under sevoflurane anesthesia
acid (TCA) solution (24% w/w) was added to the digested formulae (Morikubo Yakuhin Inc., Kanagawa, Japan). The rats were
and the mixtures were centrifuged at 12,500×g for 20 min at 20℃. sacrificed while still under anesthesia. The cecum was excised and
The nitrogen content of the supernatant (A) was determined using weighed, and the cecal contents were collected and stored at _80℃
the Dumas method (AOAC, 2005). Each test formula also received until analysis.
the same treatment but without the enzyme digestion steps and The 8 day body weight gain, total food consumption, and the
TCA precipitation, and its nitrogen content (B) was determined. nitrogen contents of the test formulae, urine and feces were used to
The degree of digestion was defined as A/B × 100 (%) calculate the following nutritional values: food efficiency ratio
In vivo studies (FER), true digestibility (TD), biological value (BV) and net
Animals Five-week-old male Wistar rats were purchased from protein utilization (NPU).
576 Y. Takeda. et al.

Blood analysis The serum albumin concentration was determined of the UHT formula. The CML contents of the retort A and retort
using a Micro Assay Albumin kit (AKJ Global Technology Co., Ltd., B formulae were 3.9 fold and 4.4 fold higher, respectively, than
Chiba, Japan). that of the UHT formula. The available lysine contents for the
Cecal analysis The nitrogen concentration in the cecal content retort A and retort B formulae were lower (11% and 24%,
was determined using the Dumas method (AOAC, 2005), while the respectively) than that for the UHT formula.
ammonia concentration was determined using an Ammonia-Test- SDS-PAGE Figure 1 shows the result of SDS-PAGE analysis.
Wako kit (Wako Pure Chemical Institute, Ltd.) (Okuda et al., The main band of the UHT formula had the same mobility as that
1965). of sodium caseinate, which is the sole protein source in the test
Statistical analysis All data are expressed as means ± SEM. formulae. The retort A formula had blurred bands with relatively
Statistical significance between the groups was determined using high molecular weights; while in the retort B formula, no strongly
an analysis of variance followed by the Tukey-Kramer method. A stained protein band was observed.
value of p < 0.05 was taken to indicate statistical significance, and In vitro protein digestibility The in vitro digestibility of the
all analyses were carried out using the JMP software package UHT formula (40.1%) was higher than that of the retort A (38.5%)
(Version 4.0; SAS Institute, Cary, NC, USA). and retort B (35.8%) formulae (Fig. 2). The high degree of
reproducibility in the result was confirmed.
Results In vivo assays
In vitro studies Body weight gain and food intake The body weight gain of
Physicochemical properties The physicochemical properties rats fed the UHT, retort A and retort B formulae during the test
of the three test formulae, including color and pH, are presented in period was 45.2 ± 1.5, 44.0 ± 1.7 and 25.9 ± 3.0 g, respectively
Table 3. Representative results are shown. Regarding the color, the (Fig. 3). The increases in the UHT and retort A groups were
“L” value of the UHT formula was higher than for the retort A and significantly greater than in the retort B group ( p < 0.01).
retort B formulae, whereas the “a” and “b” values of the UHT The food intake of rats fed the UHT, retort A and retort B
formula were lower than for both retort formulae. Moreover, for formulae during the test period was 458 ± 9, 450 ± 15 and 360 ±
the retort A formula, the “L” value was higher and the “a” and “b” 21 g, respectively. Again, food intake was significantly greater in
values were lower than for the retort B formula. These values are the UHT and retort A groups than in the retort B group ( p < 0.01).
in accordance with the appearance of the formulae. The pH of the In addition, the daily food intake of rats fed the UHT and retort A
UHT formula was higher than for both retort formulae. formulae was highly similar. And the daily food intake was
Glycation products Table 4 presents the furosine, CML and consistently lower in the retort B group than in the UHT and retort
available lysine contents of the test formulae. Representative A groups during the test period.
results are shown. The furosine contents of the retort A and B Protein digestibility and bioavailability Indices of nutritional
formulae were 3.5 fold and 3.6 fold higher, respectively, than that value are presented in Table 5. The values of FER, TD, BV and

Table 3 . Effects of sterilization processes on the color and pH value of the test formulae.

UHT formula Retort A formula Retort B formula

Appearance

Color

L value 73 . 5 67 . 9 58 . 5
a value −1 . 9 1.6 5.5
b value 9.3 13 . 3 16 . 8
pH 6.8 6.6 6.2

Table 4 . Effects of sterilization processes on the furosine, carboxymethyllysine

and available lysine contents of the test formulae .
UHT Retort A Retort B
Component
formula formula formula
Furosine (mg/100 g protein) 56 . 1 195 . 8 204 . 7
Carboxymethyllysine (mg/100 g protein) 16 . 8 65 . 2 74 . 3
Available lysine (g/100 g protein) 9.6 8.5 7.3
Effects of Sterilization on Liquid Enteral Formula Protein Quality 577

Fig. 2. Effects of sterilization processes on the in vitro digestibility.

Fig. 1. Effects of sterilization processes on protein

polymerization as shown on an SDS-PAGE gel. The
arrowhead indicates casein with a high molecular weight.

Fig. 3. Effects of test formulae produced by different

sterilization processes on weight gain in rats. Results
are expressed as means ± SE. Different letters indicate
significant differences ( p < 0.001).

NPU were highest in the UHT group, significantly greater than in

the retort B group (p < 0.001, p < 0.001, p < 0.01, and p < 0.05,
respectively). Fig. 4. Effects of test formulae produced by different sterilization
Serum albumin The serum albumin concentration of rats fed processes on cecal parameters in rats. Results are expressed as means
the UHT, retort A and retort B formulae was 41.1 ± 0.6, 42.8 ± 1.7 ± SE. A: Cecal weight of rats; B: Cecal nitrogen concentration; and
C: Cecal ammonia concentration. Different letters indicate significant
and 38.1 ± 1.0 mg/mL, respectively. There were no significant
differences (A: p < 0.001; B: p < 0.01; C: p < 0.01).
differences among the three groups.
578 Y. Takeda. et al.

Table 5 . Effects of sterilization processes on the nutritional value of the test formulae .

Nutritional value UHT formula Retort A formula Retort B formula p value

(n = 10) (n = 10) (n = 8)
FER 9 . 86 ± 0 . 15 a 9 . 78 ± 0 . 08 a 6 . 86 ± 0 . 44 b p < 0 . 001
TD 98 . 28 ± 0 . 17 a 97 . 57 ± 0 . 20 a 95 . 79 ± 0 . 38 b p < 0 . 001
BV 71 . 46 ± 1 . 06 a 69 . 61 ± 2 . 04 a b 64 . 09 ± 1 . 36 b p < 0 . 01
NPU 70 . 19 ± 1 . 02 a 67 . 92 ± 2 . 00 a 61 . 43 ± 1 . 52 b p < 0 . 05
FER, food efficiency ratio; TD, true digestibility; BV, biological value; NPU, net protein utilization . Values are means ±
SE . Values followed by different letters are significantly different .

Cecal content The cecal weight of the UHT, retort A and acidic compared to the UHT formula, while the retort B formula
retort B groups was 1.88 ± 0.1, 1.96 ± 0.1 and 2.86 ± 0.2 g, was more acidic than the retort A formula. Sweetsur and White
respectively (Fig. 4A), and was significantly higher in the retort B (1975) reported that the Maillard reaction may be involved in heat-
group than the UHT and retort A groups ( p < 0.001). The cecal induced acidification of milk. Therefore, the decrease in pH
nitrogen concentration was lowest in the UHT group, significantly observed with a longer sterilization time in this study may result
lower than in the retort A and retort B groups ( p <0.001) (Fig. 4B). from the promotion of the Maillard reaction. These results strongly
The cecal ammonia concentration displayed similar tendencies to suggest that the retort processes affect the physicochemical
the cecal nitrogen concentration, and was significantly higher in the properties of the enteral liquid formula more intensively compared
retort B group compared to the UHT and retort A groups with the UHT process.
( p < 0.001) (Fig. 4C). Protein modification We investigated three indices (furosine,
CML and available lysine) to evaluate the degree of glycation and
Discussion protein modification in the test formulae. Furosine is a compound
We observed in a preliminary study that the sensory properties formed during acid hydrolysis of Amadori compounds, which are
of an enteral formula were affected by the heat treatment used in produced by the reaction of the ε-amino group of lysine with
sterilization. It was assumed that this change can be attributed to reducing carbohydrates. It is considered a specific and early
the Maillard reaction. Therefore, we investigated whether heat indicator of the Maillard reaction (van Boekel, 1998). Modified
treatment affects not only sensory properties but also lysine produced by the Maillard reaction is nutritionally
physicochemical and nutritional properties of a liquid enteral unavailable. Therefore, available lysine, which is unmodified, is
formula. To investigate the effect of sterilization process on these used as an index of not only the Maillard reaction but also of the
properties of liquid enteral formulae, we prepared a formula and amino acid nutritional property (Goodno et al., 1981). Rufián-
sterilized it by three processes: UHT (150℃, 5 s) and two kinds of Henares et al. (2006) reported that the loss of available lysine in
retort processes (retort A: 121℃, 10 min; retort B: 121℃, 40 min). ingredients sterilized at 140℃ was considerably higher than for
These are typical processes used in the sterilization of dairy that sterilized at 100℃. Prolonged thermal treatments induced
products (Burton, 1988). The F value of the retort B process was further reactions with primary reaction products, generating
set to be similar to that of the UHT process, and the F value of the advanced glycation end products (AGEs). CML, a form of AGE,
retort A process was set to be lower than the UHT and retort B has been used as an AGE marker (Uribarri et al., 2010).
processes. The test formulae were evaluated in vitro, and in vivo In the present study, the furosine and CML contents of the
using rats. Additionally, we hypothesized that the sterilization retort A and retort B formulae were much higher than in the UHT
process might affect the intestinal environment of rats fed the test formula. It was also observed that a decrease in the available lysine
formulae and therefore investigated the cecal content of rats. content was associated with an increase in Maillard reaction
In vitro assays products (Table 4). These results indicate that both retort processes
Physicochemical properties The effect of heat treatment on accelerate protein glycation and losses in the lysine nutritional
the physicochemical properties of the test formulae was value relative to the UHT process. Recently, the relationship
investigated. In the color determination test, the “L” value of the between Maillard reaction products and health promotion/
UHT formula was higher than that of the retort A and retort B deterioration has received considerable attention. Regarding the
formulae. In contrast, the “a” and “b” values of the UHT formula health benefits of Maillard reaction products, in vitro studies have
were lower than those of the retort A and retort B formulae. These reported that Maillard reaction products may affect intestinal
results were consistent with their appearance (Table 3) and bacteria (Corzo-Martínez et al., 2012; Dominika et al., 2011).
indicated that both retort processes accelerated the Maillard Corzo-Martínez et al. (2012) reported that Maillard reaction
reaction compared with the UHT process. products derived from milk protein displayed similar functions as
The pH of the retort A and retort B formulae was moderately prebiotics, i.e., acted to increase certain intestinal bacteria including
Effects of Sterilization on Liquid Enteral Formula Protein Quality 579

lactic acid bacteria and bifidobacteria. In contrast, it was reported to the UHT formula. Burton (1988) reported in a sensory test that
that AGEs are associated with a deterioration in human health in-container milk sterilization had less flavor acceptability
(Bohlender et al., 2005; Kurz et al., 2011; Uribarri et al., 2011). compared to UHT sterilized milk. These flavor changes might
AGEs are considered as pro-oxidant substances and might play an affect the amount of food intake in rats. Additionally, pH
important role in the pathogenesis of diabetes and several other differences between formulae might affect food intake. However,
diseases, including kidney and neurological diseases. Uribarri et al. considering the FER result, the lower body weight gain observed in
(2010) proposed practical methods to reduce the dietary intake of the retort B group may be induced by the decreased nutritional
AGEs. In this context, for patients and the elderly who ingest value rather than by a reduction in food intake.
enteral formula as their sole nutrient source, it may be necessary to Further nutritional values of each formula were evaluated
reduce the amount of AGEs in enteral formula products. Further based on the nitrogen balance method (AOAC, 2005). The TD, BV
studies should be performed to clarify the relationship between and NPU values in the retort B group were significantly lower than
human health and Maillard reaction products in enteral formulae. in the UHT group (Table 5). The nutritional values in the retort A
Protein polymerization, in the context of protein modification, group were lower than in the UHT group.
was assessed using SDS-PAGE. The protein pattern of the UHT Regarding TD, the result of the animal assay was consistent
formula was similar to that of sodium caseinate. In the retort A with the in vitro digestion assay. Sarwar et al. (1989) compared
formula, a high molecular weight band (~150 kDa) was observed. commercial infant formula products in a rat study and
In the retort B formula, no clear protein band was observed, demonstrated that the powdered form exhibited superior
indicating that the casein had aggregated and was too large to enter digestibility to the liquid form. According to the manufacturer’s
the polyacrylamide-gel used in SDS-PAGE analysis. information, the heat treatment during the production of liquid
In summary, the protein included in both retort formulae was formula was more intense than that for the powdered form,
more highly glycated and polymerized than that in the UHT indicating the influence of heat treatment on digestibility. The
formula. lower BV in both retort groups compared with the UHT group
In vitro digestion We performed an in vitro digestion assay to might be related to the diminished available lysine content (Table
assess protein digestibility. The digestibility of both retort formulae 4). From the NPU result, representing the ratio of nitrogen retained
was lower than that of the UHT formula (Fig. 2). This suggests that in the body to ingested nitrogen, it was suggested that the entire
digestive enzymes could not react with the protein region adjacent protein bioavailability of the UHT group is superior to both retort
to the modified lysine, and that the aggregated protein structure groups.
inhibited its enzymatic degradation, a result that is in agreement Lacroix et al. (2006) reported that there was no difference in
with Swaisgood and Catignani (1985). Wada and Lönnerdal (2014) NPU between high temperature short time (HTST; 72℃, 20 s or
reported that sterilization, such as UHT and in-can sterilization, 96℃, 5 s) pasteurized skimmed milk and UHT (140℃, 5 s)
might improve the milk protein digestibility of raw milk, and that sterilized skimmed milk. These results suggest that the heating
in-can sterilized milk showed better protein digestibility compared temperature (72 _ 140℃) does not significantly affect the
with UHT processed milk. The differences in protein digestibility nutritional value of milk protein when only a relatively short heat
might be attributable to differences in the test materials. treatment time is utilized.
In vivo assays The results of the present study suggest that the retort A or B
Nutritional evaluation The results of the in vitro assays process (long time and relatively low temperature) induces lower
presented above showed that heat induced protein modification, protein quality compared with the UHT process (short time and
including glycation and polymerization, may affect the nutritional relatively high temperature); nevertheless, the F value of the UHT
properties of proteins. process was higher than that of both retort processes.
In our preliminary study, rats were fed the UHT formula or a Serum albumin The serum albumin concentration, which is an
purified rodent diet (AIN-93G) to confirm the suitability of the index of systemic nutritional status and in particular protein
enteral liquid test formula. The weight gain of rats fed the UHT nutritional status (Pain et al., 1978), did not differ significantly
formula was similar to those fed AIN-93G (data not shown), among the three groups. Bleiberg-Daniel et al. (1990) investigated
suggesting that the UHT formula contained sufficient nutrients for the nutritional status of rats fed a diet deficient in tryptophan, an
normal rat growth. essential amino acid, during an 8-day test period. They reported
Our animal study demonstrated that the body weight gain of that there was no difference in the albumin concentration between
the retort B group was significantly less than that of the UHT and the control and tryptophan deficient rats, despite a loss of body
retort A groups (Fig. 3). The food intake and the FER of the retort weight in the tryptophan deficient animals. They suggested that the
B group were also significantly lower than those of the UHT or albumin concentration was influenced by a more severe protein
retort A group (Table 5). The lower food intake observed in the deficiency.
retort B group might be attributable to flavor differences compared Taken together, the amount and quality of protein of the three
580 Y. Takeda. et al.

test formulae in the present study were sufficient to maintain basal the application of the appropriate process to liquid enteral
metabolism. The protein in the retort B formula may be used formulae.
preferentially when it is necessary to maintain the systemic
nutritional condition, with growth as the priority. References
Cecal analysis The cecal weight of the retort B group was AOAC, (2005). AOAC, Official Methods of Analysis of AOAC
significantly higher than in the UHT and retort A groups International (18th ed.), AOAC International, Gaithersburg, MD, USA.
( p < 0.001) (Fig. 4A). Concurrently, it was observed that the cecal Asadullah, Khair-un-nisa, Tarar, O.M., Ali, S.A., Jamil, K., and Begum, A.
nitrogen concentration, which is derived mainly from ingested (2010). Study to evaluate the impact of heat treatment on water soluble
protein, of both retort groups was significantly higher than in the vitamins in milk. J. Pak. Med. Assoc. 60, 909-912.
UHT group ( p < 0.001) (Fig. 4B). The concentrations of ammonia, Bleiberg-Daniel, F., le Moullac, B., Maire, J.C., and Wade, S. (1990).
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