Protection of cottonseed oil from in vitro rumenhydrogenation with various formaldehyde-treated proteins

Item Type text; Thesis-Reproduction (electronic)

Authors Sherrill, Donna Davies, 1943-

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this materialis made possible by the University Libraries, University of Arizona.Further transmission, reproduction or presentation (such aspublic display or performance) of protected items is prohibitedexcept with permission of the author.

Download date 24/05/2018 03:28:14

Link to Item http://hdl.handle.net/10150/554658

PROTECTION OF COTTONSEED OIL FROM IN VITRO RUMEN HYDROGENATION WITH VARIOUS

FORMALDEHYDE-TREATED PROTEINS

"byDonna Davies Sherrill

A Thesis Submitted to the Faculty of theDEPARTMENT OF ANIMAL SCIENCE

In Partial Fulfillment of the Requirements For the Degree ofMASTER OF SCIENCE

In the Graduate CollegeTHE UNIVERSITY OF ARIZONA

STATEMENT BY AUTHOR

This thesis has been submitted in partial fulfill­ment of requirements for an advanced degree at The Univer­sity of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library 0

Brief quotations from this thesis are allowable without special permissions provided that accurate acknowl­edgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his judgment the proposed use of the material is in the inter­ests of scholarship. In all other instances, however0 permission must be obtained from the author.

APPROVAL BY THESIS DIRECTOR This thesis has been approved on the date shown belows

’ WILLIAM H, HALE'.: Date' Professor of Animal Science

ACKNOWLEDGMENTS

The author wishes to express her sincere appreciation to her major professor* Dr„ William H„ Hale* for his con­tinued guidance and constructive criticism throughout her graduate program and for his assistance in the design and implementation of studies reported in this thesis0 A spe­cial thank you is extended to Doctors C„ B. Theurer* Fo D„ Dryden and W„ C„ Brown for their assistance throughout the course of this study, Sincere appreciation is also ex­tended to Doctors Bo L„ Reid and C 0 W 0 Weber of the Poultry Science Department for their assistance in the application of statistical procedures* The author wishes to express her gratitude to the staff and graduate students of the Animal Science Department for their encouragement and assistance*

A very special thanks to my husband * Lewis* for his patience* understanding and support and to my family whose continued help and encouragement made this program possible*

iii

)TABLE OF CONTENTS

PageLIST OF TABLES 0 0 * 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 viABSTRACT O O O O O O O O O O O O O O O O O O O O O VliINTRODUCTION o o o o o o o q o - 0 0 0 0 00 0 0 0 0 1LITERATURE REVIEW 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 4

Protein Protection from Rumen Degradation . . 5Crosslinking of Proteins with Formaldehyde . . . 8Protection of Fats from Rumen Hydrogenation . 10Digestion and Metabolism of Formaldehyde-

Treated" Products o o 0 o 0 o o 0 o 0 o o 0 o 15MATERIALS AND METHODS . . » . o . 0 0 » 0 • . 18

General 0 0 0 0 0 o o o . o o o o o o o o o o 18Homogenization» Formaldehyde Treatment andInc ubatxon . . . . o . . . o . . . . 0 0 . 18

Analytical Procedures . . . . . . . . . . . . 21Lipid Extraction and Percent Lipid

Determination 0 0 0 0 0 0 . 0 0 0 0 . 21Fatty Acid Analysis . . . . . . . . . . . 22Statistical Treatment of Data . . . . . . 26

RESULTS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 27In Vitro Fermentation of Casein-Cottonseed

Oil Emulsion O O O O O O O C O . O O O O O 27In Vitro Fermentation of Collagen (m. w 0 1,000)-Cottonseed Oil Emulsion . . 0 0 . . 0 . . 30

In Vitro Fermentation of Collagen (m. w .10.000)-Cottonseed Oil Emulsion . . . . . - 33

In Vitro Fermentation of Collagen (m. w.200.000)-Cottonseed Oil Emulsion . . . . . 36

In Vitro Fermentation of Isolated Soy Protein-Cottonseed Oil Emulsion . . . . . . . . . 39

GENERAL DISCUSSION . . . . . . . . 0 . . . . . . . 42SUMMARY O O O O O O O O O O O O O O O O O O O O O " 46

iv

TABLE OF CONTENTS— Continuedv

PageAPPENDIX As ANALYSIS OF VARIANCE . . . . . . . . . 49LITERATURE CITED . . . . . . . . . . . . . . . . . 51

Table1.

2o

3o

to

5 =

60

LIST OF TABLES

The effect of in vitro fermentation with rumen fluid on the hydrogenation of the unsaturated fatty acids in a casein- cottonseed oil emulsion 0 0 „ „ * „The effect of in vitro fermentation with rumen fluid on the hydrogenation of the : unsaturated fatty acids in a collagen (m. Wo 19000)-cottonseed oil emulsioh „ 0 <The effect of in vitro fermentation with rumen fluid on the hydrogenation of the unsaturated fatty acids in a collagen (me Wo 10p 000)-cottonseed oil emulsion , 0 ,The effect of in vitro fermentation with rumen fluid on the hydrogenation of the unsaturated fatty acids in a collagen (m0 Wo 200p000)- cottonseed oil emulsron o o o o o o o o o o o o o o o o tThe effect of in vitro fermentation with rumen fluid on the hydrogenation of the unsaturated fatty acids in an isolated soy protein-cottonseed oil emulsion 0 0 0 » « <Analysis of variance for means of stearicp oleic and linoleic acids in protein- cottonseed oil emulsions „ o <> „ „ <, <, o <

Page

2§

31

34

3?

40

50

vi

ABSTRACT

Various proteins were emulsified with cottonseed oil . and treated with formaldehyde to inhibit microbial hydroge­nation of the polyunsaturated fatty acids during in vitro fermentation with rumen fluid0 Five different proteins were useds casein, isolated soy protein and three collagens with molecular weights (m6 w 0) of 1,000, 10,000 and 200,000, Formaldehyde additions were 0 „ 75^» lo50fo and 3° 00$ by weight of the protein. In addition the emulsions were divided into three sub-groups $ undried, treated with formaldehyde prior to drying and dried prior to treating with formaldehyde0 Samples were incubated in vitro with rumen fluid for 20 hourso

Drying the emulsion either before or after formalde­hyde treatment was the most effective method of protecting the unsaturated fatty acids of cottonseed oil from hydroge­nation, Although formaldehyde treatment of the undried product afforded some protection, particularly for the col­loid (m, w, 10,000) emulsion treated with 3o00$ formalde­hyde, in general the protection was not complete,

All levels of formaldehyde effectively protected the casein-cottonseed oil emulsion treated prior to drying. Protection of the 1,000 m, w, collagen was incomplete in all of the samples regardless of drying treatment or .

vii

viiiformaldehyde level. The 10„000 and 200,000 m, w, collagens were equally well protected with 3,00^ formaldehyde applied prior to or after drying. Protection of the isolated soy protein-cottonseed oil emulsion was incomplete for all drying treatments and all levels of formaldehyde application.

INTRODUCTION

It is an accepted practice in commercial feedlots of the western United States to add fat at levels of y to 4# of the rations of fattening cattle« The primary reason for fat additions is to increase the caloric density of the ration without lowering the roughage content„ Fat in the diet of ruminants is subject to hydrolysis and hydrogenation of the unsaturated fatty acids by microorganisms in the rumen (Carton, 196l)» If more than 4% fat is added to rations, there is a marked increase in excretion of insoluble fecal soaps and a decrease in digestibility of crude fiber (Figroid, 1971). Soluble soaps that are formed with ele­ments such as potassium tend to act as bactericidal agents, complexing with the protein of the microbial cell walls thus inhibiting microbial activity in the rumen (Valko,1946? Hotchkiss, 1946)„

At times the price of inedible tallow is such that it competes favorably with grain as an energy source and addi­tions of more than 4% would be desirable. Therefore, it would appear that if the amount of fat added to cattle rations is to be increased, the fat will have to be pro­tected from rumen microbial attack so that it can bypass the rumen and be digested as in the monogastric animal.

Due to hydrogenation of the dietary unsaturated fats by rumen microorganisms„ beef depot fat is highly saturated when compared to fish and poultry fat. For this reason con­sumption of high levels of meat from beef has been ques­tioned by the American Medical Association® The reason for this is that the medical profession believes there is a re­lationship between saturated fat and certain cardiovascular diseases®

Treatment of proteins such as casein with formalde­hyde or suitable agents will result in prevention of protein hydrolysis by rumen microorganisms (Hughes and Williams* 1971)- Chemically the formaldehyde primarily crosslinks with the epsilon amino group of lysine (French and Edsall* 19^6)0 The crosslinkage is stable at the normal pH levels (5-7) in the rumen® When the formaldehyde linked protein passes into the abomasum with its low pH (2-4) the cross- linkage is broken® The protein is then subject to the usual enzymatic hydrolysis in the abomasum and small intestine®

Scott* Cook and Mills (1971) demonstrated that the unsaturated fatty acid content of depot fat in steers and lambs could be increased by feeding a spray dried emulsion of casein and polyunsaturated fat treated with formaldehyde® The unsaturated fat was protected from rumen fermentation due to the fact that the fat particles were surrounded by the formaldehyde treated protein®

In the studies reported to date casein has served as the emulsifying protein. However, casein is expensive and not readily available.

This study was conducted to find a suitable protein to emulsify with fat and the most effective level of formalde­hyde to protect the product from rumen degradation. Unsat­urated fat was used to serve as an internal tracer to deter­mine the degree of protection. Five different proteins were homogenized with cottonseed oil and treated with three levels of formaldehyde then incubated in vitro with rumen fluid and analyzed to evaluate the amount of protection of polyunsaturated fats from hydrogenation. The proteins used were casein, collagen (molecular weight (m, w,) 1,000), Swift's Colloid IV (m, w, 10,000), Swift's Colloid 5V (m, w,200,000) and isolated soy protein from the Drackett Products Company, In addition the treated homogenates were incubated undried, treated with formaldehyde prior to drying and dried prior to treating with formaldehyde.

LITERATURE REVIEW

It is a well known fact that fats fed in the diet to ruminant animals are hydrolyzed by rumen microorganisms and most of the unsaturated fatty acids are hydrogenated in the rumen and pass on to the remainder of the digestive tract as saturated fats (Garton, 1961; and Ward, Scott and.Dawson* 1964)0 It is also well substantiated that soluble dietary proteins are hydrolyzed and deaminated by rumen microorga­nisms resulting in high ammonia production and carbon chains used for microbial growth (McDonald* 1952)° As a result the primary protein available to the ruminant animal for diges­tion is microbial protein (McDonald, 1954). Chalmers* Cuthbertson and Synge (1954) have demonstrated that placing soluble proteins directly into the abomasum or duodenum of sheep increased nitrogen retention and wool growth when com­pared to sheep receiving the same proteins in their ration.

Since the studies by Hegsted et al. (1965) which showed the effects of high levels of saturated fatty acids on human serum cholesterol concentrations* there has been considerable interest in increasing unsaturated depot fat in beef cattle.

4

Protein Protection from.Rumen Degradation It is a generally recognized fact that ruminants

utilize high quality proteins inefficiently due to microbial degradation of dietary protein in the rumen, Cuthbertson and Chalmers (1950) reported greater nitrogen retention when casein was infused into the duodenum than when it was intro­duced into the rumen, McDonald (1954) and McDonald and Hall (1957) calculated that 90% of casein fed was converted to microbial protein,

Chalmers et al, (1954) and Reis and Schinkel (1963 and 1964) showed that nitrogen retention and wool growth were enhanced by abomasa! or duodenal infusion of proteins. The studies of Reis and Schinkel (1964) indicated that the sulfur amino acids in casein administered duodenally were very efficiently utilized by the wool follicles and that the amount of sulfur amino acids absorbed from the intestine is a primary factor influencing rate of wool growth,

Chalmers (1961) states that the ideal ration for max­imum use of nitrogen should contain protein of good digest­ibility with a low solubility in the rumen. This assumption, is based on the premise that high rumen ammonia values are indicative of poor nitrogen utilization. It is obvious that an adequate amount of nitrogen must be supplied for the growth and activities of rumen microorganisms; however, any excess degradation of high quality proteins would be wasteful.

Hatfield (1970) presented evidence to support the hypothesis that pattern and levels of essential amino acids reaching the absorption sites are among the first perfor­mance limiting factors in the non-lactating ruminant. He concluded that the practical and realistic method of supple­mentation would be with a protein protected from rumen al­teration, suggesting a pH sensitive coating on the protein as a possible method.

There are three obvious methods of getting dietary protein into the abomasum according to Ferguson, Hemsley and Reis (1967) without reduction in its subsequent utilization by the host. These methods are; (1) reduction of the time spent by the protein in the rumen, (2) reduction of the pro­tease and/or deaminase activity of rumen microorganisms, and (3) protection of dietary protein from microbial attack in the rumen. Protection may be achieved by chemical and phys­ical modification of the protein by coating individual par­ticles with a protective envelope, or by a combination of these methods. However, any modification would have to leave the protein in a form that would allow for digestion and absorption in the post-nominal tract,

Leroy and Zelter which was reported by Ferguson et al, (1967) successfully decreased rumen degradation of peanut and soybean protein using tannins, Chalmers et al, (195^) found that heat-treatment of casein reduced ammonia forma­tion and decreased the rate of breakdown in the rumen

7resulting in increased nitrogen utilization in sheep.Tagari et al. (1965) also claimed success by treating pro­teins with vegetable tannins. Research reports indicate that the nutritional value of tannin and heat treated pro­teins may be impaired (Danke et al., 1966). Hatfield (1970)used tannic acid treated dietary soybean meal on feedlot

)steers and successfully improved performance (average daily gain and nitrogen retention)=

Several workers are investigating the feasibility of a pH sensitive coating which would render the protein insolu­ble at the rumen pH of approximately six but which would break down in the abomasum which has a pH of about three. Ferguson et al. (196?) treated casein with a 4% solution of formaldehyde and added it to a control diet fed to Merino sheep. The sheep fed treated casein showed no increase in rumen ammonia, a 70$ increase in wool growth' and a substan-

• tial weight increase over the control group. They deter­mined that at least 80$ of the nitrogen in the treated ca­sein was digested and absorbed after leaving the rumen, but only 4$ was degraded in the rumen.

Reis and Tunks (1970) measured the changes in plasma amino acids in sheep receiving no casein, untreated casein, formaldehyde treated casein (4$) and untreated casein in­fused into the abomasum. Treated casein in the diet and casein per abomasum caused similar changes in the propor­tions of amino acids in the plasma and was most evident in

that the amount of essential amino acids increased, Addi- tion of untreated casein to the diet caused very little change in plasma amino acids when compared to the control,

Similar results have been reported by Hughes and Williams (1971) and Offer, Evans and Axford (1971)= Several reviews are available on protein protection studies (Ward @t el,, 1964 and Hatfield, 1970)=

Crosslinking of Proteins with Formaldehyde The purpose of formaldehyde treatment to a protein is

to render it insoluble to microbial attack in the rumen.The formaldehyde produces crosslinkages which result in an increase in the molecular weight of the original protein (Nitschmann, Hadom and Lavener, 19^§)« The resultant prod­uct has a reduced elasticity and is relatively insoluble at a pH of five and above, but the crosslinkage is broken at a pH of three (Walker, 1964),

There are numerous possible reactions of formaldehyde with different proteins and the available literature is plentiful, French and Edsall (1946) present a comprehensive review of the possible linkages involved in combining form­aldehyde with various proteins and amino acids® Both col­lagen and casein are discussed in some detail®

The lysine portion of the protein is the probable site of crosslinkage with formaldehyde (French and Edsall, 1946g and Walker, 1964)® French and Edsall (1946) report that

each of the two amino groups of lysine should be capable ofreacting with formaldehydee but the epsilon amino group ap­pears to be the most active„ Studies of the action of form­aldehyde on collagen and deaminated collagen by Gustavson . (19^0) as reported by Walker (1964) support the hypothesis that the formation of methylene cross-linkages in this pro­tein is dependent upon the presence of the epsilon amino groups of the lysine residue»

The pH of the reaction media is a controlling factor in the action of formaldehyde on proteins in an aqueous mediao However, its effect is dependent on the type of re­active groups available in the protein involved and the reaction temperature (Walker, 1964) In most cases, the rate of reaction increases with increasing temperature (Lumiere, Lumiere and Seyewetz, 1906) as reported by Walker (1964)0 In commercial tanning of leather the amount of formaldehyde which reacts is less on the acid side of the isoelectric points however, some proteins differ in this respect (Walker, 1964)0 Results indicate that formaldehyde is fixed on the epsilon nitrogen atoms of the lysine radicals of collagen at pH 6.-8 (Gustavson, 1940)6

Practically all artificial protein fibers are given a stabilizing treatment with formaldehyde in the course of manufacture (Atwood, 1940)e This procedure confers water resistance, strength and protection from biological attack,, At present the majority of these fibers are produced from

10casein and zein, but soybean and peanut proteins are also of commercial interest and considerable research has been car­ried out with other proteins (Walker, 1964)0

Protection of Fats from Rumen Hydrogenation Scott et al® (1971) described a method whereby linseed,

safflower and sunflower oil droplets were protected from in vitro rumen hydrogenation by encapsulation with formaldehyde treated protein® This was accomplished by emulsifying ca­sein, oil and water in a colloid mill, homogenizing the , . . emulsion in a two-stage homogenizer and then spray drying the homogenate® The oil-casein particles were treated with 4 to^5^ formaldehyde either prior to drying or by spraying after drying® The most complete protection was obtained with the product containing a final concentration of 20lj6 formaldehyde on a protein basis® Linoleic acid was com­pletely protected after 20 hours of in vitro incubation with sheep rumen fluid. When lower concentrations of formalde­hyde were used, the majority of the unsaturated fatty acids were still protected, whereas 90% of the untreated linoleic acid was hydrogenated. Scott and coworkers also found that varying the type of oil and the oil-protein ratio (Isl, Zsl, 3:1) had no significant effect on hydrogenation of the oil in the treated emulsions®

Scott et al. (1970) incubated formaldehyde treated linseed oil-casein particles (1:1) in vitro with sheep rumen

11fluid for 0, 8 and 20 hours» The treated particles were completely protected from hydrogenation and the linoleic and linolenic acid values were the same before and after incu­bation.

In vivo studies by Scott, et al. (1970) showed that formaldehyde treated linseed oil-casein particles (Isl) were protected to the same extent as in their in vitro studies0 Examination of abomasal contents showed a marked increase in dienoic and trienoie acids of goats on the diet containing treated particles over those receiving untreated particles. In fact the proportions of linoleic and linolenic acids observed in abomasal contents of animals fed the untreated supplement were very similar to those of animals on a basal diet receiving no supplement.

Cook et al. (1970) fed 8 to 10 week old lambs on a diet containing formaldehyde, protected casein-safflower oil particles (Isl) for three and six weeks. After slaughter it was determined that there was a three to five fold increase in the proportion of linoleic acid in the depot fats of the experimental animals over the control. The fatty acid changes were similar for lambs slaughtered at three weeks and six weeks. These researchers concluded that it is pos­sible to produce polyunsaturated fats in ruminants by feed­ing formaldehyde treated protein-oil particles for only a few weeks prior to slaughter.

12Tove and Mochrie (1963) reported that when polyunsat­

urated oils were directly infused into the circulatory sys­tem of ruminants, there was a substantial incorporation of the constituent fatty acids into the plasma and milk glyc­erides. Although this method is not practical for dairy use, it shows that the fatty acid composition of milk can be altered,

Scott et al„ (1970) and Scott and Cook (1970) studied the effect on the fatty acid profile of milk from goats fed a diet containing formaldehyde treated linseed oil-casein particles (Isl)„ These workers observed a 20/& increase in the proportion of linolenic acid in the milk from animals on the treated diet and an increase of only Jlfo on the untreated diet over the control animals.

Using the same diet as above, except with 2 si oil- protein particles, fed to milk cows, Scott et al, (1971) were able to report significant increases in the proportions of linoleic and linolenic acids in the milk fat. These re­sponses were observed within 24 to 48 hours after inclusion of the protected supplement to the diet.

Plowman et al, (1972) used the protein-fat protection method devised by Scott et al, (1971) with safflower oil and casein (1,5*1). This product was treated with 2$> form­aldehyde (on a protein basis) and samples were incubated with rumen fluid to determine the extent of protection of unsaturated fatty acids from hydrogenation. The protein

13oil complex was completely protected after 24 hours of incu­bation. However6 after 48 hours of incubation approximately 50% of the linoleic acid was converted to either oleic or stearic acid. This indicates that the oil was protected for the major part of the expected transit time in the rumen (Hungate, 1966).

When Plowman et al0 (1972) fed the above product to dairy cows at a rate of 1500 grams per day there were no significant changes in the amount of milk produced» but there was an increase of 1 to 1,5% in the fat content of the milk. The increase in fat content was noted within 24 hours after the protected product was introduced into the diet and persisted for 24 to 48 hours after removal of the product from the diet. There was also a rapid increase of linoleic acid concentration from a standard of 3 to 4% to a high of 30 to 35% of the total milk fatty acids.

Cook, Scott and Pan (1972) described the effects of feeding formaldehyde protected casein-safflower oil (Isl) particles on the fatty acid composition of milk and plasma lipids with three breeds of dairy cows. Feeding the treated supplement at a rate of one kilogram per head per day caused substantial increases in the proportions of lino­leic acid in triglycerides of both plasma and milk from all three breeds of cows. These increases were associated with decreases in the proportions of myristic, palmitic and to a lesser extent oleic acid. When the formaldehyde treated

14supplement was replaced by an equivalent amount of untreated protein-oil supplement there were marked decreases in the proportions of linoleic acid and corresponding increases of oleic acid in both the milk and plasma triglycerides» There was no consistent effect of feeding either treated or un­treated supplements on the proportions of stearic acid in the plasma and milk lipids0

Scott et ale (1972) developed two separate procedures for supplementing casein and safflower oil to lactating goat rationso Sodium hydroxide» sodium caseinate, water, lecithin and formaldehyde were homogenized in one procedure and spray-dried as described by Scott et al, (1971)0 This mixture had a total solids content of approximately 20%„The second mixture contained the same ingredients in differ­ent proportions with a final total solids content of approx­imately 40 to 45%0 The second mixture was fed as a gel rather than spray-drying. The fatty acid composition of the milk was analyzed during a three week feeding trial0 Milk from the animals on the protected supplement showed an in­crease in the proportion of linoleic acid from 3 to 40 to 20 to 30^0 The response to the gel supplement was greater than to the dried supplement but the authors felt that the difference was related to the higher intake of oil by the goats consuming the gel0

15Digestion and Metabolism of Formaldehyde^

Treated ProductsHogan, Connell and Mills (1973) determined the diges­

tion of protected casein-safflower oil particles in a lu­cerne hay ration as compared to a straight lucerne hay rationo Sheep fitted with cannulae in the rumenp abomasum and terminal ileum were used for the experiment„ These workers reported significant increases in the flow of di­gests from the abomasum but no effect on the volume of water in the rumen or on the rates of flow from the rumen or ter­minal ileum„ The fact that the movement of the digests through the rumen was not effected supports the conclusions that there is little digestion of the particles there0 The increased flow from the abomasum with the treated diet was probably associated with the beginning of digestion of the formaldehyde treated particles and the return to normal of the flow rate past the terminal ileum is consistent with the observation that the components of the lipid supplement had been previously absorbed in the small intestine«

Hogan et al, (197 3) calculated that a diet composed . of lucerne hay plus 20% protected casein-safflower oil par­ticles supplies about 30% more metabolizable energy„ 38% more net energy and 70% more amino acids than lucerne hay alone <>

A study was conducted by Macrae et al0 (1972) to measure the disappearance of nitrogen and amino acids from

16different regions of the digestive tract of sheep given dietary supplements of formaldehyde treated casein0 The sheep were fitted with cannulae in the rumen, duodenum and ileumo These researchers reported a net retention of sup­plementary nitrogen of 36# for treated casein as opposed to 17% with untreated casein. Daily amounts of non-ammonia nitrogen entering the small intestine were increased (P<oQl) as were the amounts of non-ammonia nitrogen appar­ently absorbed therein (P < o05), Apparent absorption of amino acids from the small intestine was greater (P <,05) , with treated casein0

'Faichney* Cook, :Scott and. Davies in:19?2 studied VJ?- the composition of plasma, liver, muscle and adipose tissue lipids in cattle fed treated and untreated casein-safflower oil supplements. These researchers reported an increase (P < 0G5) in the proportion of linoleie acid in the plasma triglycerides of growing cattle fed the treated supplement. They observed a substantial increase in the triglycerides of depot fats and muscle but the rate of increase was less than that noted in the plasma triglycerides. However, the proportion of linoleie acid in liver triglycerides was simi­lar to that in plasma0

Mills et al« (1972) investigated the metabolism of formaldehyde when given to ruminants as an aldehyde-casein- oil complex0 They used the spray dried safflower oil- easein (1;1) complex described by Scott et alc (1971)

17treated with 1.5% formaldehyde on a protein basis. This supplement was added to the diet of sheep and lactating goats for two, six and eighteen months. The results showed that sheep effectively metabolize formaldehyde when it is fed in an aldehyde-casein-oil complex. The metabolism ap= pears to be influenced by length of reaction time prior to - - feeding. In all of the experiments a large proportion of ingested formaldehyde was metabolized to methane and carbon dioxide. They discovered no resultant accumulation of form­aldehyde in the tissues or milk of any of the test animals.

A review of the literature indicates that an effective and practical means of protecting fat from microbial degra­dation would have a tremendous effect on the ruminant live­stock industry.

The availability of poly-unsaturated ruminant meats and dairy products; would be of value in the diets of pa­tients with cardiovascular diseases.

MATERIALS AND METHODS

GeneralFive experiments were conducted using cottonseed oil

homogenized with five different proteins and treated with three different levels of formaldehyde to determine the de­gree of protection of the unsaturated fatty acids from rumen hydrogenation in vitro, The treated groups were further divided into various subgroupss undried versus dried homogenate, formaldehyde treatment applied prior to drying, and treatment applied after drying.

The homogenization was carried out in the Dairy and Food Sciences Laboratory, In vitro fermentation with rumen fluid was conducted at the University of Arizona Campbell Av­enue Farm and the lipids were extracted and chromatographed in the Meat Sciences Laboratory of the Animal Science Department,

The proteins used were casein,, commercial grade collar gen {molecular weight (m, w,) 10000j from Oscar Meyer» isola­ted soy protein from Drackett Products Company, collagen (m, % 10,000) and collagen (to, w, 200,000) from Swift and Company,

Homogenization, Formaldehyde Treatment and Incubation

Each protein was hydrated for 12 hours at a pH of 6,5 with three to four parts water prior to homogenization. The

protein and cottonseed oil were then heated with constant stirring to a temperature of 60 C in a steam kettle0 In all homogenates the ratio of cottonseed oil to protein was 70s30 except in the case of isolated soy protein when the ratio was 60:40. The heated mixture was passed through a Manton Gaulin Laboratory homogenizer Model 15M-8TBA,, two stage valve body assembly equipped with a two horsepower, 1800 RPM motor, stainless steel tank, three way valve and bypass assembly with dampener, at 3s000 lbs, pressure. To assure complete homogenization, the mixture was passed through the homogenizer twice. The casein and collagens (m, w, 10,000 and 200,000) emulsions contained approximately 50$ water while the collagen (m, w„ 1,000) emulsion con­tained 36% water and the isolated soy protein emulsion con­tained 62% water.

The homogenate was divided into two parts. One part of the homogenate was separated into three equal portions and mixed with three different levels of formaldehyde ex­pressed as a percent of protein weight (0,75$, 1=50% and 3-00%). The source of formaldehyde was 37% formalin.

Each of these treatment groups was divided1 one half was not dried while the remainder was dried in a vacuum oven at 50 C for 72 hours.

The second part was further divided into four groups. One fourth was kept as a control and was stored separately from the treated samples. The other three groups were

20dried and then treated with the three levels of formaldehyde shown above. Schematically the treatment groups were as follows I

Homogenate

Wet

Wet DryWet Dry Wet Dry

After all treatment and drying procedures were accom­plished, duplicates of each sample were weighed into 125 ml. Erlenmeyer flasks in amounts to yield approximately .60 grams of cottonseed oil per flask.

Rumen contents were removed from a fistulated Here­ford steer fed alfalfa hay only. The rumen contents were strained through four layers of cheesecloth into a pre­warmed one gallon Thermos jug. The fluid was centrifuged for 10 minutes at 2500 RPM to remove the remaining hay particles.

Fifty ml. of rumen fluid was added to each Erlenmeyer flask. The flasks were placed into a water bath at 37 C and carbon dioxide was bubbled at a slow rate through the samples during the incubation period of 20 hours. The

21primary purpose of the carbon dioxide was to keep the samples stirred.

Analytical Procedures

Lipid Extraction and Percent Lipid DeterminationFollowing incubationg the flasks were taken to the

Meat Sciences Laboratory for the lipid extraction. Lipid extraction was a modification of the chloroform-methanol procedure of Brown (1969)e and the procedure followed is listed below,

1, Put each sample in a 300 ml, stainless steel Omni- Mixer jar with 3 mlo of concentrated hydrochloric acid,'

2. Add 100 ml, methanol and mix at high speed (speed control ± 6,0) for 3 minutes,

3o Remove sample from mixer, add 100 ml, of chloro­form and mix again for 3 minutes at high speed,

4, Filter through two pieces of Van Lab coarse (#28306-10) filter paper in a Buchner funnel. Wet filter / paper with distilled water prior to filtering,

5, After sample has completely filtered, wash mixing jar with 10 - 15 ml, of chloroform and transfer contents to funnel,

6, Transfer filtrate to 250 ml, graduated cylinder. Rinse flask with approximately 10 ml, of chloroform, add to cylinder and stir well. To the unincubated controls add an additional 40 ml, of distilled water and stir.

227o Allow samples to sit overnight in the graduated

cylinder so that the two phases clearly separate»8 e Read and record the volume of the lower phase

(chloroform and fat)» Remove the upper phase by suction and discard =,

9» Remove a 20 ml, aliquot from the lower phase and place in a predried and analytically weighed 50 ml, beaker. Discard remainder of lower phase,

10, Evaporate contents of beakers to dryness by leaving them in a vacuum oven overnight at 50 C,

11, One hour prior to removal, increaseevacuum and then remove samples and obtain dried weight analytically,

12, Bring lipid back in solution with a small amount (approximately 2 ml,) of chloroform and transfer to a labeled two dram vial. Rinse beaker with chloroform and add to vial,

13o Calculationsvolume of CHClo layer

* (dry) * 100All extractions were run in duplicate.

Fatty Acid AnalysisThe extracted lipids were transesterified as described

by Cramer and Marchello (1964), The modified procedure was as followss

23Ip Place 9 or 10 drops of lipid in a clean, dry, 15 ,

ml, centrifuge tube and add 2 ml» of benzene»20 Add 4 ml, of 0.5# sulfuric acid in methanol (by

volume), add 2 - 3 boiling stones and cap tubes tightly»3o Place tube in a 90 C water bath for 2.5 hours and

stir sample frequently.4, Remove tube and cool.5o Wash solution with 8 - 10 ml. of demineralized

water? then add 2 ml. of redistilled petroleum ether0 Mix well o

6o Centrifuge solution for 10 minutes at 1500 RPM07. Transfer all of top phase (petroleum ether layer)

into a 4 ml. screw cap vial.8. Add 2 ml. redistilled petroleum ether and repeat

centrifugation.9» Transfer off top phase and add to vial.10. Store in freezer (-18 C) until the thin layer

chromotography procedure.A thin layer chromotography procedure was used to

separate non-fatty acid lipid components from the sample after the methylesters were formed and before introduction of the sample into the gas-liquid chromatograph.

The thin layer chromatography procedure was as follows 8

1. Evaporate sample with nitrogen until dry.

242» Take samples up in 1 or 2 ml. chloroform and ,

transfer to 10 ml. centrifuge tubes8 rinse vials with 1 or 2 ml. chloroform and add to appropriate tubes.

3» Dry samples with nitrogen until only a drop or two remains in the tube bottom. Add about 0.03 ml. of chloro­form with a micro-syringe to each tube and shake to remove sample from the sides of the tube.

4. Prepare plates (Silplate - F~220 Brinkmann- Instruments,, Inc.) by measuring and scraping division lines on the plate so that it is divided into three equal columns. This allows three samples to be spotted on each plate.

5o Dry plates in 130 C oven for one hour9 then desic­cate for 45 minutes. .

6. Spot plates by applying the sample on a thin line with 150 lambda pipet across the plate (about 4 cm. from bottom),

7<> Place spotted plates in the solvent vat containing 200 ml, pentane. 25 ml, di-ethyl ether, 2.5 ml. glacial ace­tic acid until solvent migration has reached the top of the plate.

8. Remove plates from solvent vat and spray with a 0,05% solution of Rhodamine 6g in ethanol.

9. With a razor blade remove the visible fatty acid band on each plate which had migrated a similar distance to a methyl-palmitate fatty acid standard.

25lOo Put each band in a separately numbered centrifuge

tube and add about 5 ml0 of pentane, cap tubes and shake011o Centrifuge tubes for five minutes at 2*000 RPM012, Remove upper phase (pentane layer) and put into

two dram vials,13, Repeat steps 10* 11 and 12 only use ether in place

of pentane,14, Store in freezer (-18 C) until gas liquid chroma­

tographic analysis (within 7 days),15, Remove sample from freezer and completely evapo­

rate solvents with a stream of nitrogen in a 50 C water bath. Redilute with known volume of chloroform for injec­tion into the gas liquid chromatograph,

16, To store sample after a portion has been analyzed* add 1,0 ml, of pentane and replace in freezer,

A Hamilton 1,0 microliter syringe was used for the introduction of sample into the gas-liquid chromatograph, Each sample injected ranged from 0,1 to 0,3 microliter.The esters were chromatographically separated using a Beck­man GC-5 instrument equipped with a flame ionization detec­tor, A 1,83 m, (3,2 mm, 0, D,) coiled stainless steel column packed with 100 to 120 mesh chromosorb (HP) coated with 15% of diethylene glycol succinate was used for the fatty acid separations. Instrument operating conditions weres column temperature* 197 Gg inlet temperature * 205 Gg

26detector line temperatureg 240 C ; detector temperature„260 C 1 and carrier gas flow (nitrogen)» 28 ml./minute.

Identification of the fatty acid methyl esters was accomplished by comparison of the relative retention times with those of standard solutions of known compositions. The weight percent of each ester was determined by computing its proportionate amount, as measured by a disc integrator, to the total area of the chromatogram.

Statistical Treatment of DataAnalysis of variance and Duncan°s new multiple range

test were used to analyse the data (Steel and Torrie, I960), Those effects found to be significant were at P <,05 level. The primary consideration was for 18 carbon fatty acids to determine the degree of treatment protection from hydroge­nation.

Analysis was run within each of the 018 fatty acids comparing all of the sample means within that acid regard­less of treatment. Analysis was not run between different acids or the same acids and different trials.

Two samples were run for each treatment and a mean was computed for analysis.

RESULTS

In Vitro Fermentation of Casein-CottonseedS=SS ~̂ )'TTT5iau 1 slon

Incubation of the untreated samples resulted in a marked reduction (20%) in the linoleic acid (018*2) content when compared to the non-incubated control (Table 1)„ These differences were not significant. The oleic acid (018*1) and stearic acid (018*0) values for the incubated untreated samples were higher (P< .05) than the control. As 018*2 was hydrogenated/ an increase in 018*1 and 018*0 was to be ex­pected, The relatively low level of hydrogenation of 018*2 in the incubated untreated sample was probably due to limy ited microbial activity during incubation. Significant re­duction in the 018*2 content of the incubated untreated sam­ples was achieved in subsequent trials reported in this manuscript, Very little change was noted in the myristie acid (014*0), palmitic acid (016*0) or palmitoleic acid (016*1) for any of the treatments in this trial.

The 018*2 values for the wet, treated samples were not lower (P > .05) than the unincubated control except for the sample treated at the 3.00% level of formaldehyde, The 018*2 values for all undried, treated samples were not dif­ferent (P < . 05) from the untreated, incubated control. If formaldehyde treatment had offered complete protection, the

2?

Table 10 The effect of in vitro fomentation with rumen fluid on the hydrogenationof the unsaturated fatty acids in a casein-cottonseed oil emulsion.*

Formaldehyde Treatment Percent Fatty Acids

014 Cl6 Cl6tl C18 s0 018 $ 1 C18 32Control Emulsion - Not Inuebated Wet Emulsion - Incubated

0,9 25.3 1,1 1.7a 18.8a 52,2b

None 1,0 22.3 1.1 7,6® 24,6b 43,lab0.75% 1,3 28.1 1.1 4,7cd 20,0a 44,7ab1,50$ 1.1 22.8 0,9 3.8*° 24,4b 46,9ab3,oo$ 1,3 26,9 0,9 4.4*° 20.1* 41,3*

Dry Emulsion .<= IncubatedNone 0.9 22,5 0.7 6„0de 34,4b 43,0ab

Treated Prior to Drying0.75$ 1.4 22.5 0.7 3.0** 20.9* 51 = 51.50$ 0.9 22,3 0.8 2.3** 20,6* 52,6b3,00$ 0.9 22,8 0,8 3.2a*° 20,4* 51,8ab

*Means of duplicate samples.abcde^eang within the same column with unlike superscripts are different

(P< .05) o

29value should have been significantly higher than the incu­bated control» The Cl8 $l content of the undried samples treated with 0,75 and 3<>00% formaldehyde were lower (P <«05) than the incubated control, but they were not different (P x,05) from the non-ineubated control. The Cl8;l for the undried sample treated with 1,50% formaldehyde was higher (P <,05) than the non-incubated. control. The C18 $0 values for the undried samples at all formaldehyde treatment levels was different from both the incubated and unincubated controls. Since the treated samples were not significantly different (P >,05) from the incubated control, protection was not complete.

All three formaldehyde treatment levels for the sam­ples treated prior to drying were essentially 100% effective in protection of C18;2 from hydrogenation. The values were essentially the same as the non-incubated control. The ClSsl and 018:0 values for these samples were not signifi­cantly different (P >.05) from the unincubated control but lower (P <„05) than the incubated control.

The results of this trial indicated that formaldehyde treatment more effectively protected the dried samples than it did the undried samples,

30In Vitro Fermentation of Collagen {ifu w 0 1,000)-

""Cottonseed Oil Emulsion .Incubation of the untreated emulsions, wet or dried,

resulted in a marked reduction (P <,05) of the linoleic acid content from the non-incubated control value (Table 2)„This indicated that the in vitro fermentation was effective0 The corresponding increase in Cl8sl and C18$0 was higher (P < o 05) for these two samples than the non-incubated con­trol, It can also be seen in Table 2 that there were small increases in the Cl6«0 values for all incubated samples 0 Very little change was noted in the C1*H0 and Cl6*l acid values for any of the samples.

The Cl8i2 values for all of the treated samples were higher (P <„05) than the incubated control values. However, the wet emulsion treated with 1,50% formaldehyde and the sample treated with ).00% formaldehyde prior to drying were the only two treatments in this trial which were not sig­nificantly lower (P >.05) than the non-incubated control,

The C18s2, C18il and C18$0 values for the wet emulsion treated with 0.75% formaldehyde were different (P <,05) from both the incubated and unincubated controls indicating that there was some protection from hydrogenation but it was not complete. The C18:1 and 018:0 values for the undried sam­ples treated with 1,50% and 3®00% formaldehyde were not sig­nificantly different (P >,05) from the non-incubated control

Table 20 The effect of in vitro fermentation with rumen fluid on the hydrogenationof the unsaturated fatty acids in a collagen (m0 w 6 leOOO)-cottonseedoil emulsion,*

Formaldehyde Treatment Percent Fatty Acids

014 016 C16 si Cl8s0 ClSsl C18 s2Control - Not Incubated 1,1 22,0 1,2 ' 3,8a 21, la 50.3dWet Emulsion - Incubated

}•$6°X&hc

None0,75%1,50%3,00%

0,91,00,91,0

23.923.9 24,4 24,6

0,90,80,71,0

32.8?

21, la

27.6a

45.4°Dry Emulsion - Incubated

ll.lef 31.0bcNone 1.3 24,7 0.4 29,8aTreated Prior to Drying0.75%1,50%3,00%

1,11,01,0

24,724.523.6

0,70.61,1 21.5a

Dried Prior to Treating

&

0.75%1,50%3.00%

1,01,01,0

25.924.325.3

1,00.90.7 1:1. 44.1°

*Means of duplicate samples,abcdefMe^ng same columns with unlike superscripts are different

(P <,05),

32but they were significantly lower (P <.05) than the incu­bated control.

None of the Cl8sl values reported for the samplestreated prior to drying or dried prior to treating were dif­ferent (P >.05) from the non-incubated control. However, the sample dried prior to treating with 3•00$ formaldehyde was the only one of the dried samples which did not have a higher (P >.05) CI81O value than the non-incubated control although all of the dried sample values for CI81O were lower (P <.05) than the values for the incubated controls. Values for all of the 18 carbon fatty acids reported in Table 2 for the dried emulsions show a 10$ or greater decrease in the Cl8s2 fatty acid value and a 10$ or greater increase in theClSfO fatty acid value for each sample when compared to thenon-incubated control.

In this trial it appears that formaldehyde treatment afforded the best protection from hydrogenation at the 1.50$ level for the undried samples and at the 3®00$ level treated prior to drying for the dried samples. In general all lev-_ els of formaldehyde treatment afforded some protection for the dried samples when compared to the untreated, dried control, however, protection was not complete when compared to the non-incubated control.

In Yitro Fermentation of Collagen (m. w„ 10,000)- ' Cottonseed.OTT^Emul'sion"

Incubation of the untreated emulsions, both wet and undried, resulted in a marked decrease (P < =05) in the C18 s2 content and a corresponding increase (P <=05) in the 018*1 and 018*0 fatty acids when compared to the unincubated con­trol values. Essentially no change was noted in the 014*0, Cl'6s0 and 016*1 values for the incubated untreated samples (Table 3)= .

The 018*2 of the wet emulsion treated with 0,75^ form­aldehyde was extensively hydrogenated and the values for 018*2 and 018*1 were not significantly different (P >,05) from the incubated control although the value for 018*0 was lower (P <,05), The 018*2 and 018*0 levels for the undried samples treated with 1,50% formaldehyde were different (P <,05) from both the incubated and unincubated controls indicating partial hydrogenation of the unsaturated fatty acids. Almost complete protection was afforded by treatment of the undried sample with 3 <>00% formaldehyde as indicated by the non-significant change (P >,05) in 018*2 and 018*1 as compared to the unincubated control,

Linoleic and oleic acid values for the samples treated with 0,75% and 3.00% formaldehyde prior to drying were dif­ferent ( P < ,05) from both incubated and unincubated controls Stearic acid values were lower (P c 0 05) than the incubated untreated sample but were not different (P >,05) from the

Table 3« The effect of in vitro fermentation with rumen fluid on the hydrogenationof the unsaturated fatty acids in a collagen (m. w. 10,000)-cottonseedoil emulsion.*

Formaldehyde Treatment Percent Fatty Acids

Control - Not IncubatedWet Emulsion - Incubated

None 0.75%1.50%3.00%

Dry Emulsion - IncubatedNone

Treated Prior to Drying0.75#1.50#3.00#

Dried Prior to Treating0.75#1.50#3.oo#

C14

0.91.01.10.9

0.91.50.9

0.90.81.9

C160.6 20.9

20.621.121.420.3

1.1 21.3

33.6 34.521.7

20.519.921.0

Cl6 ;l0.8

1.51.20.71.4

0.9

0.80.70.9

0.70.91.1

018:02.5'

C18;1 C18 12,cd24.0'

8r | l3.7de 33.4

1:0:a

3-7b=de22.3̂ c3:M%abc

54.1'

33.1ab

I;:.37.3

43.3°42.0=55.3

49.552.951.9

dede

*Means of duplicate samples, abcdef

(P <.05). Means within the same column with unlike superscripts are different

35unincu'bated control0 However, there was a large increase in the palmitic acid values for these two samples. These changes in Cl6 s0 did not occur in any of the other trials reported except the trial with collagen m, w„ 200,000, Protection of the sample treated with 3°00$ formaldehyde prior to drying was complete in that the C18i2 and C18;0 values were not different (P >,05) from the unincubated control and the C18i1 value was slightly lower than the same control.

As with the other treatments discussed in this trial, 0,75$ formaldehyde did not completely protect the sample treated after drying, Linoleic and oleic acid values were different (P< , 05) from both incubated and unincubated con­trols although the stearic acid value for this sample was the same as the dry incubated control. Protection for the samples dried prior to treating with 1,50$ and 3*00$ form­aldehyde was excellent. There was no difference (P >.05) between the Cl8s2 and Cl8sl values for these two samples when compared to the non-incubated control. There was a slight rise in the CI81O values for these two samples which was significantly different (P <,05) from the non-incubated control but not from the incubated dried control (P >,05).

Results from this trial indicated that the best pro­tection from hydrogenation was achieved with 3.00$ form­aldehyde treatment of all three emulsions and with 1,50$ formaldehyde applied after drying.

36In Vitro Fermentation of Collagen (m. w. 200,000)-

^cottonseed Oil Emulsion "Hydrogenation of the undried and dried incubated

controls was extensive as shown in Table 4. A decrease of 48% and 46%, respectively, was noted for C18:2, an increase of 46% and 45% in ClSsl content and an increase of 58% and 55% in 018*0 fatty acid was noted for the undried and dried incubated controls, respectively, as compared to the non­incubated control values. All of the C18*2, 018*1 and 018*0 fatty acid values for these two samples were different ( P < ,05) from the non-incubated control.

There was incomplete protection of the wet emulsion at all three levels of formaldehyde treatment„ Values for the three 18 carbon fatty acids were different (P <,05) from both incubated undried and non-incubated controls. There was no change in the 014*0, 016*0 and 016*1 values for the undried treated samples, but an increase was noted in the 014*0 and 016*0 fatty acids of the incubated control when compared to the unincubated control,

Incomplete protection was also noted for 018*2 and 018*1 fatty acids in samples treated with 0,75% and 1,50% formaldehyde prior to drying. The 018*2 and 018*1 values for these two samples were different (P <,05) from both the dried incubated control and the non-incubated control. However, the 018*0 values for these two samples were not

Table 4. The effect of in vitro fermentation with rumen fluid on the hydrogenationof the unsaturated fatty acids in a collagen (m, w. 200,000)-cottonseedoil emulsion.*

Formaldehyde Treatment Percent Fatty Acids

C14 C16 Cl6; 1 018*0 C18.1 C1812Control - Not Incubated 1.1 21.0 1.0 2,2a 19.5b 54.0fWet Emulsion - Incubated

None 2.3 25.8 1.2 5.2* 35.5g 28.3*0.75# 0.9 20.9 1.0 tP 8:1 39.9b1.50# 1.0 20.4 1.2 44.1°3.oo# 1.0 20.9 0.7 23.9a 48.2*

Dry Emulsion - IncubatedNone 2.1 26.1 0.9 4.8° 35.1 29.2*

Treated Prior to Drying0.75# 0.9 20.4 1.1 3.7^ 24.2* 48.4*

49.2*'1.50# 1.1 20.8 1.6 %;3bcd 21.3°3.00# 1.4 23.4 1.0 13.6* 52.81Dried Prior to Treating0.75# 0.9 20.8 0.9 2:̂ 50.1?1.50# 0.9 20.0 0.5 54.413.00# 1.2 20.1 0.9 3.4b 19.3 53.01

*Means of duplicate samples.abcdefMeans within the same column with unlike superscripts are different

(P< .05).

38significantly different (P >,05) from the dried incubated control although they were higher (P<,05) than the non­incubated control.

The 018$2 value of the sample treated with 3,00% formaldehyde prior to drying was well protected from hydro­genation and reflected no significant difference (P >=05) from the non-incubated control 018$2 value. Oleic and stearic acid values for the 3°00% treatment prior to drying were different (P <,05) from the non-incubated control» however, the mean differences were small.

Drying prior to treatment with 0,75% formaldehyde resulted in a significant decrease (P <,05) in the unsatu­rated 0188 2 fatty acid as compared to unincubated control, however, the percentage decrease was small indicating a good degree of protection. This was accompanied by an in­crease (P<,05) in 0188l and 018:0* Drying prior to treat­ment with 1,50% and 3®00% formaldehyde was effective in protecting the fatty acids from hydrogenation. There was no significant difference (P >,05) between the 018s2 and 018il values reported for these two levels and the non- incubated control, Only a slight but significant increase (P <»05) was noted for the 018:0 values over the non- incubated control.

Results from this trial indicated that formaldehyde treatment was not effective at any of the three levels of formaldehyde tested with the undried emulsion. Excellent

39protection from hydrogenation of 018 82 was achieved with 3o00% formaldehyde treatment prior to drying as well as . .. 1,50% and 3,00% treatment applied after drying.

In Vitro Fermentation of Isolated Soy Protein- B===:===== CotTonseed Oil kmulsion

Hydrogenation of the 018i2 fatty acids in both the wet and dried incubated untreated samples was extensive liable 5)° The 018s2e Cl8sl and Cl8s0 content of these two samples were different (P<,05) from the non-incubated control.

As noted in previous trials with other proteins0 treatment of the wet emulsion with 0,75% formaldehyde did not provide good protection from hydrogenation of the 018*2 fatty acid although it was different (P <,05) from the in­cubated control indicating some degree of protection. An increase was noted in 018*1 and 018*0 levels for the 0,75% formaldehyde treated undried sample which was significantly different (P <,05) from the non-incubated and incubated control.

The linoleic, oleic and stearic acid values reported for the undried samples treated with 1,50% and 3=00% form­aldehyde were similar. However6 the 018*2 and 018*0 values were different (P <,05) from both the non-incubated and in­cubated controls. It can be seen from Table 5 that the values reported for the three 018 fatty acids reflect less than a 10% change from the non-incubated control samples

Table 5* The effect of in vitro fermentation with rumen fluid on the hydrogenationof the unsaturated fatty acids in an isolated soy protein-cottonseed oilemulsion.*

Formaldehyde Treatment Percent Fatty Acids

C14 C16 Cl6il CI81O ClSil C18i2Control - Not Incubated 1.2 21.9 1.1 3.9a 20.9ab 50.5f.Wet Emulsion - Incubated SNone

0.75%1.50%3.00%

1.01.00.91.0

24.024.0 24.4 24.6

0.90.80.71.1

32.8°

1 1Dry Emulsion - Incubated

13.01None 1.5 24.2 0.6 32.1° 29.4°Treated Prior to Drying0.75%1.50%3.00%

1.11.01.0

24.7 24.423.8

0.70.71.2 ft!:. E l i

Dried Prior to Treating0.75%1.50%3.00%

1.01.11.0

26.024.325.4

1.01.00.7

7:7p4i:lt 5.6 25.8°

44.2^

♦Means of duplicate samples.abcdefghiMeans within the same column with unlike superscripts are different

(P <.05).

41indicating a relatively high degree of protection although it was not complete0

The level of formaldehyde did not appear to effect the hydrogenation of the three samples treated prior to drying and dried prior to treating. There was a 10^ de­crease in the Cl8s2 content of the three treatment groups which was different (P<,05) from the unincubated control. No significant difference (P >,05) was noted for the C18si values reported for these three treatments and the unincu­bated control* but the C18s0 values were higher (PC,05) than the unincubated control and lower ( P C ,05) than the incubated control.

Relatively little difference was noted between the 18 carbon fatty acid values for the three formaldehyde treatment levels of samples treated prior to drying and those dried prior to treating in this trial.

In this trial none of the drying treatments or form­aldehyde levels used provided complete protection of the cottonseed oil from hydrogenation.

GENERAL DISCUSSION

The protein-fat emulsions incubated without formalde­hyde treatment were well hydrogenated and reflected a 50$ reduction in linoleic acid for four of the five trials0 With the casein-fat emulsion there was an 18$ decrease in the C18s2 acid. These results indicate an active fermenta­tion of the unprotected fats in the in vitro system.

In general protection of the undried samples was incomplete with one exception. The undried collagen [molecular weight (m. w.) 10,0003-cottonseed oil emulsion treated with 3.00$ formaldehyde was completely protected from hydrogenation. As there was some protection afforded by formaldehyde treatment of the undried samples for all of the proteins used except casein, additional studies should be conducted with undried treated emulsions. These trials represent only one incubation for each sample even though they were duplicated within treatment and thus do not definitely establish that the undried samples were not pro­tected, Scott et al. (1971) successfully protected a ca- sein-safflower oil gel in in vivo studies with goats and noted a response similar to that achieved with the spray- dried emulsion. It has not been established what effect moisture content of the emulsions and formaldehyde levels greater than 3®00$ may have on hydrogenation of incubated

4-2

43samples„ Most of the reports reviewed to date (Scott et aL, 1971 . and Plowman et al<,0 1972) have been with dried emul­sions of casein and polyunsaturated fats.

For the casein-cottonseed oil emulsion treated prior to drying9 complete protection of the 018:2 fatty acid was achieved at all levels of formaldehyde treatment (0.75»1.50 and 3=00$)» Scott et al. (1971) reported complete protection for a casein-safflower oil emulsion treated with 4.8$ formaldehyde (on a protein basis) prior to drying. Lower levels (1.6 and 3=2$) were not completely effective in their studies. In this series of trials formaldehyde treatment following drying was not studied with casein- cottonseed oil emulsion. Scott et al. (1971) reported that -the fat-protein ratio (1:1, 2:1, 3*1) of the emulsions and the type of fat used (safflower, sunflower and linseed oils) had no effect on the degree of protection achieved.

With the collagens and isolated soy protein, treatment with 3=00$ formaldehyde resulted in similar CIS:2 values within a protein, whether treatment was applied prior to or after drying. Application of 3.00$ formaldehyde completely protected both of the high molecular collagens when applied prior to and after drying. Application of 1,50$ formalde­hyde was completely effective in protecting C18:2 acid for two collagens (m, w, 10,000 and 200,000) dried prior to treating but not for samples treated prior to drying. With the exception of the latter samples, the application of

#1«50^ formaldehyde did not effectively protect the dried emulsions„ The application of 0,75% formaldehyde was not effective on any of the emulsions except the dried casein producto ,

Collagen m„ w« lc000 and isolated soy protein (used for encapsulation of the cottonseed oil) were less effec­tive in preventing hydrogenation of the polyunsaturated fat as compared to the other proteins0 Since there have been no prior reports on these two proteins for fat encap­sulation, there is no evidence to support or refute the protein effect0 The reason for the difference in level of protection afforded by the different proteins is not understood.

There are many factors involved in the reaction of formaldehyde with proteins and future research may provide reasons for the results found in these trials. In these studies all emulsions were carried out at the same tempera­ture and pH, Work done by Gustavson (1940) as reported by Walker (1964) shows that formaldehyde is fixed on the ep­silon nitrogen atoms of the lysine radicals of collagen at pH 6 - 8 and on both lysine and arginine at pH 12„ In addition the formaldehyde effect is dependent on the type of reactive groups available in the protein. Collagen is high in proline and hydroxyproline which do not crosslink due to an absence of free amino groups, Collagen is low in

45lysine when compared to casein and is therefore low in ly­sine amino sites for crosslinkage with formaldehyde„

The results of this study indicate that polyunsatu­rated fats can "be protected from rumen microbial hydrogena­tion by encapsulation with formaldehyde treated protein and that proteins other than casein may be effective encapsu­lating agents. In general 3•00^ formaldehyde appeared to be the most effective treatment level for complete protec­tion of the dried emulsions with the exception of collagen (m, w„ 1,000) and isolated soy protein0 Results from these trials also indicate that emulsions dried either prior to or after formaldehyde treatment were more effectively pro­tected from microbial hydrogenation„ Additional research into the crosslinkage mechanisms involved with formaldehyde and different proteins and the effect of pH and temperature should be studied more thoroughly 0

SUMMARY

This study was conducted to find a suitable protein to emulsify with cottonseed oil and the most effective level of formaldehyde to protect the emulsion from rumen microbial hydrogenation of the polyunsaturated fatty acidso Five different proteins were emulsified with cottonseed oil with three different drying treatments# undried» treated with formaldehyde prior to and after drying0 Three levels of formaldehyde (0,75, 1,50 and 3=00%) were applied to each drying treatment prior to in vitro incubation with rumen fluid, ■

After 20 hours of incubation the lipid was extracted from the samples, the fatty acids esterified and the fatty acid profile was determined by gas liquid chromatography.

In the casein-cottonseed oil emulsion results indi­cated that formaldehyde more effectively protected the dried samples than it did the undried samples. In general this was found to be true throughout the entire series of trials.

In the trial using commercial grade collagen fmolecu^-, lar weight (m, w,) 1,,0005-cottonseed oil emulsion formalde­hyde treatment at the 1,50$ level for the undried samples and at the 3* 00$ level for the samples treated prior to drying provided the most effective protection from microbial

46

47hydrogenation of the Cl8s2 acid. However, even with these two formaldehyde levels the linoleic acid was not cornel pletely protected.

With the collagen (m, w, 10,000)-cottonseed oil emulsion, treatment with 3*00% formaldehyde afforded ex­cellent protection for the undried emulsion as well as the two dried emulsions. The 1,50/S formaldehyde effectively protected the emulsion treated prior to drying. Most complete protection was obtained for the sample treated with JoOQfa formaldehyde prior to drying.

The results from the trial with collagen (m, w,200,000)-cottonseed oil emulsion agreed with the results from the trial with collagen (m, w, 10,000) except that none of the undried samples were well protected. Complete protection was obtained with the 3,000 formaldehyde treat­ment of the emulsion prior to drying and with the samples dried prior to treatment with 1,500 and 3,000 formaldehyde.

Results from the trial using isolated soy protein- cottonseed oil indicated that emulsions were not completely protected from microbial hydrogenation with any drying treatment or any level of formaldehyde treatment,

In general the most effective protection from hydro­genation was afforded with 3,000 formaldehyde treatment of the samples either prior to or after drying. Formaldehyde treatment of wet emulsions was not completely effective at

48any of the levels tested with the exception of collagen (ra. Wo 108000) as noted above0

Caseiq, collagen (m. w 0 10,000) and collagen (mo w„200,000) were the most effective encapsulating proteins used in this trial0

APPENDIX A

ANALYSIS OF VARIANCE

49

Table 6„ Analysis of variance for means of stearic* oleic and linoleic acids in protein-cottonseed oil emulsions„

Proteins Source of Variation d o f o Mean Squares

Stearic - Oleic LinoleicCasein

Among Treatments Within Treatments

89

6.532**.#3

15.696**1.393

42,321**18,304

Collagen (m. w. 1*000) Among Treatments Within Treatments

1112

18.102**1.26?

46.599**1.776

132,231**2,576

Collagen (m. w, 10*000) Among Treatments Within Treatments

1112

1.111.152 92.391**4,692 120,718**

2,338Collagen (m. w. 200*000)

Among Treatments Within Treatments

1112

1.481.240 95.164**

,298164,250**

.724Isolated Soy Protein

Among Treatments Within Treatments

1112

17.100**,204

42,180**.747

100,206** . 944

**(P< .01)

Vxo

LITERATURE CITED

Atwoodg F. C . 19^0. Natural protein-base fibers, Ind,Eng, Chem, 32s154?,

Brown, J, 1969. The influence of bovine muscle stimula­tion on fat deposition, M, S, Thesis, University of Arizona, Tucson,

Chalmers, Margaret, 1961, Protein synthesis in the rumen.In Digestive Physiology and Nutrition of the Ruminant, D, Lewis (Ed,) Butterworths, London.

Chalmers, Margaret, D, P. Cuthbertson and R, L. M, Synge, 1954. Ruminal ammonia formation in relation to the protein requirement of sheep. Duodenal administration and heat processing as factors influencing fate of casein supplements, . J. of Agri/ Sci, 44s255»

Cook, L . J ., T, W, Scott, G. J, Faichney and H. L. Davies.1972. Fatty acid interrelationships in plasma, liverB muscle and adipose tissues of cattle fed safflower oil protected from ruminal hydrogenation. Lipids 7 s83,

Cook, L, J,, T, W, Scott,: K, A, Ferguson 'and I, W, McDonald,. 1970. Production of polyunsaturated ruminant body fats. Nature 228s178,

Cook L, J,, T, W, Scott and Y, S. Pan, 1972, Formaldehyde treated casein safflower oil supplement for dairy cows, II. Effect on the fatty acid composition of plasma and milk lipids, J, Dairy Res, 39*211,

Cramer, D, A, and J, A, Marchello, 1964, Seasonal and sex patterns in fat composition of growing lambs,J, Animal Sci, 23*1002,

Cuthbertson, S. P. and M. I, Chalmers, 1950, Utilization of casein supplement administered by ruminal and duodenal fistulae, Biochem, J, 46*17,

Danke, R, J =, L, B. Sherrod, E, C„ Nelson and A, D. Tillman, 19660 Effects of autoclaving and steaming of cotton­seed meal for different lengths of time on nitrogen solubility and retention in sheep, J, Animal Sci,25 * .18.1.

.51

52Faichney, G. J ,, H„ L. Davies„ T, W. Scott and L, J. Cook0

1972« The incorporation of linoleic acid into the tissues of growing steers offered a dietary supple­ment of formaldehyde treated casein-safflower oil,Aust«, J„ Biol, Sci, 25 3 205,

Ferguson, K. A,, J, A, Hemsley and P, J. Reis. 1967°Nutrition and wool growth, Aust. J. Sci. 30*215.

Figroid, Wayne C. 1971. The effect of energy intakelevel on the digestibility of high energy rations by cattle. PhD Dissertation, University of Arizona, Tucson, Arizona.

French, Dexter, and John T. Edsall, 1946. Formaldehyde with amino acids and proteins. Advances in Protein Chemistry 2*277-335» Academic Press, Inc., New York.

Garton, G, A. 1961, Influence of the rumen on thedigestion and metabolism of lipids. Digestive Phys­iology and Nutrition of the Ruminants„ Butterworths, London, England.

Gustavson, K. H, 1940, Formaldehyde reactions with pro­teins. Svensk, Kem, Tid,, 52*261,

Hatfield, E, E. 1970. Achieving the potential of highquality dietary protein for ruminants. Feedstuffs, 42*52,

.Hegsted, D, M,, R, B. McGandy, M, L, Meyers and F, J, Stare. 1965. Quantitative effects of dietary fat on serum cholesterol in man, Amer, J. Clin. Nutr. 17*281.

Hogan, J, P., P. J, Connell, S. C, Mills, 1973= The diges­tion of safflower oil - casein particles protected against ruminal hydrogenation in sheep, Aust, J,Agric. Res. 23, 87-95=

Hotchkiss, Rollin D, 1946. The nature of the bactericidalaction of surface active agents, Annals N. Y. Acad, Sci. 46*479.

Hungate, R. E. 1966, The rumen and its microbes. AcademicPress, New York, N. Y. p. 531.

Hughes, J. G . and G. L. Williams. 1971. The effect of formaldehyde treatment of protein supplements upon their in vitro fermentation and utilization by sheep. Proceedings of the Nutrition Society, 30, 41A.

53Lumiere, A., L. Lumiere and A. Seyewetz. 1906, Sur 1°

insolubilisation de la gelative par la formaldehyde<, Bulletin De La Societe Chimique De Paris. 3rd Ser. 35*872.

Macrae, J. C .0 M. J. Ulyatt» P. D. Pearce and Jane Hendtlass. 1972b Quantitative intestinal digestion of nitrogen in sheep given formaldehyde-treated and untreated casein supplements. Br. J. Nutr. 27, 39°

McDonald, I. W. 1952. The role of ammonia in ruminal digestion of protein. Biochem. J. 51*86.

McDonald, I. W. 195^. The extent of conversion of food protein to microbial protein in the rumen of sheep. Biochem. J. 56*120.

McDonald, I. W. and R, J. Hall. 1957° The conversion ofcasein into microbial proteins in the rumen. Biochem. J. 67:400,

Mills, S. C,,'L. F, Sharry, L. J, Cook and T, W. Scott.I972, Metabolism of formaldehyde when fed to rumi­nants as an aldehyde-casein-oil complex, Aust. J, Biol. Sci. 25, 807-16.

Nitschmann, H., H. Hadofn and H. Lavener. 1943,. Quantita­tive study of the binding of CH^O in the hardening of casein with CHgO sin. and with gaseous CH?0„ Hely, Chem. Acta, 26, 1069.

Offer, N. W,, R, A, Evans and R. F, E. Axford. 1971. The protection of dietary protein by formaldehyde treat­ment and its effect on the composition of duodenal digest in sheep.- Proceedings of The Nutrition Society. 30, 41A.

Plowman, R, D., J, Bitman, C. H. Gordon, L, P. Dryden,H, K. Goering, L. F. Edmonson, R. A. Yoncoskie and F. W, Douglas, Jr. 1972. Milk fat with increased polyunsaturated fatty acids, J. Dairy Sci, 55:204.

Reis, P. J, and P. G. Schinkel. 1963, Some effects ofsulfur containing amino acids on the growth and compo­sition of wool, Aust, J. Biol. Sci. 16, 218,

Reis, P. J, and P. G, Schinkel, 1964. The growth and com-- position of wool. II, The Effect of Casein, Gelatin and Sulfur Containing a. a. Gluen Per Abomasum, Aust. J, Biol. Sci, 179 532.

54Reis, P. J. and D. A. Tunks. 1970. Changes in plasma

amino acid patterns in sheep associated with supple­ments of casein and formaldehyde-treated casein.Aust. J. Biol. Sci. 23*673.

Scott, T. W., P. J. Bready, A. J. Royal and L. J. Cook.1972. Oil seed supplements for the production of polyunsaturated ruminant milk fat. Search. 3*5.

Scott, T. W. and L. J. Cook. 1970. Polyunsaturated milk fat* A New Development from Australia. Agric. Sci. Review 4th Quarter.

Scott, T. V/., L. J, Cook, K, A. Ferguson and I. W. McDonald. 1970. Production of polyunsaturated milk fat in domestic ruminants. Aust. J. Sci. 32*291.

Scott, T. W., L. J. Cook,and S. C. Mills. 1971. Protection of dietary polyunsaturated fatty acids against micro­bial hydrogenation in ruminants. J. Amer. Oil Chem­ists Soc. 48:358.

Steel, R. G. and J. H, Torrie. i960. Principles and pro­cedures of statistics. McGraw-Hill Book Co., New York.

Tagari, H., Y. Henis, M. Tamir and R. Yolcani. 1965.Effect of carob pod extract on cellulolysis, proteo­lysis, deamination and protein biosynthesis in an artificial rumen. Appl. Microbiol. 13, 437.

Tove, S. B. and R. D. Mochrie. 1963. Effect of dietary and injected fat on the fatty acid composition of bovine depot fat and milk fat. J. Dairy Sci. 46:686.

Valko, E. I. 1946. Surface active agents in biology and medicine. Annals N. Y. Acad. Sci. 46:451.

Walker, Joseph. 1964, Formaldehyde. 3rd Ed., New York, Reinhold Publ. Corp.Ward, P. V. F., T. W. Scott and R. M. Dawson. 1964. The

hydrogenation of unsaturated fatty acids in the ovine digestive tract. Biochem. J. 92*60.